A method for extracting the core of a spheroid-like biopolymer aggregate

Abstract

The application relates to a method for extracting the core of a spheroid-like biological aggregate, and relates to the technical field of sewage biological treatment, which comprises the following steps: collecting and processing a sample, separating the core, and collecting the sample; the mixed solution is collected by using a centrifugal tube through a sampling port in the reactor uniform mixing stage, and is washed three times by using deionized water after sedimentation. The core separation operation device of the biological aggregate is fixed by a lower rectangular organic glass plate as a bottom plate, and the upper layer is formed by four same four-prism organic glass blocks which are spliced towards each other; after the biological aggregate is fixed at the center of the device by using a through needle, different inner diameter capillary glass tubes are penetrated to extract and separate the cores of biological aggregates with different particle sizes; after the cores of the biological aggregates are separated, high-throughput sequencing and other analysis and research can be carried out. The application can effectively extract the cores of spheroid-like biological aggregates, and has the advantages of strong applicability, convenient and simple operation and the like.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of sewage biological treatment, and relates to a method for extracting the core of a biological aggregate, in particular, a method for extracting the core of a spherical-like biological aggregate. BACKGROUND

[0002] With the rapid development of China's economy, the scale of urban construction is expanding, and the level of urbanization is increasing. The sewage treatment capacity in China must be matched with the sewage discharge. At present, most sewage treatment plants use activated sludge method to treat sewage. However, during the operation process, it is found that the sludge production is high, the land occupation is large, and the sludge bulking is easy to occur.

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

[0004] Aerobic granular sludge has the advantages of large sludge biomass, fast settling velocity, rich microbial species, etc. In recent years, it has attracted attention. Compared with traditional activated sludge, it has the advantages of reducing land occupation, compact structure, and microbial flora partition colonization. It can effectively improve the separation of sludge and water, remove pollutants, reduce the amount of excess sludge, and reduce energy consumption. It can simultaneously remove organic matter and denitrify and dephosphorize, and is considered as one of the promising wastewater biological treatment technologies. Current research shows that the outer layer of aerobic granular sludge is mainly composed of ordinary heterotrophic microorganisms, and the outer layer structure is conducive to the supply of oxygen and the participation in aerobic metabolism. Research shows that the main factors that help promote sludge granulation and maintain stable operation of granular sludge are slow-growing bacteria: polyphosphate bacteria PAOs, polysaccharide bacteria GAOs, nitrite bacteria AOB and nitrobacteria NOB, etc. By enriching these slow-growing bacteria, stable aerobic granular sludge can be cultivated.

[0005] Anaerobic granular sludge is a self-agglomerate composed of methanogens, acetogens and hydrolysis and fermentation bacteria, which has good sedimentation performance and methanogenic activity, regular spherical structure, high specific methanogenic rate, etc. Anaerobic granular sludge is also an aggregate of various microorganisms, mainly composed of various acid-producing bacteria and methanogenic bacteria. Acid-producing bacteria are on the outside of the granule, and methanogenic bacteria are inside the granular sludge. The anaerobic bacteria in the granular sludge that participate in the decomposition of complex organic matter and the generation of methane can be divided into the following three types: the first type is hydrolysis and fermentation bacteria, which initially decompose organic matter to generate organic acid and alcohol. The second type is acetogenic bacteria, which further decompose and utilize organic acid and alcohol. The third type is methanogenic bacteria, which convert acetate and other simple compounds into methane.

[0006] The inner core of granular sludge generally refers to the core structure within the granule, which can be composed of different microorganisms. During the formation of granular sludge, the concept of the inner core is related to various formation mechanisms. For example, the crystal nucleus hypothesis suggests that the formation of granular sludge is similar to the crystallization process. Inoculum sludge or inorganic salts precipitated during reactor operation or inert organic matter serve as crystal nuclei. Granular sludge develops on the basis of the crystal nuclei and eventually forms mature granular sludge. The internal microbial community structure of granules is crucial for sludge treatment efficiency. However, to apply granular sludge technology to actual wastewater treatment plants, it is essential to understand the internal and external microbial community structure and distribution characteristics in the early stages of research. The distribution of microbial communities in the inner and outer layers of granular sludge with different particle sizes is significantly different.

[0007] Granule inner 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 internal microstructure of aerobic granular sludge, identify the redox environment in different regions, and understand how it affects the composition and function of microbial communities. In addition, granule inner core separation helps evaluate the impact of different operating conditions on the stability of aerobic granular sludge, thereby optimizing wastewater treatment process parameters, improving treatment efficiency, and enhancing the system's resistance to shock loads. For anaerobic granular sludge, granule inner core separation is also important because it can reveal the formation process, internal structural characteristics, and microbial interactions of anaerobic granular sludge. By analyzing the separated inner core and peripheral parts, we can understand the stability and functional changes of anaerobic granular sludge under different treatment conditions, such as methane production efficiency and organic pollutant degradation efficiency. This is of great significance for designing and improving anaerobic digestion processes, improving energy recovery efficiency, and reducing environmental pollution.

[0008] In actual wastewater treatment processes, there is a lack of an effective means to extract the inner core of granular aggregates due to their small particle size and relatively low particle strength. The inner core structure of granular sludge has a significant impact on its overall performance. For example, aerobic granular sludge with a strengthened inner core and skeleton can maintain better stability during long-term operation. Even if the granule breaks, the inner core can be reused to help the system recover faster. In summary, the inner core is the core structure of spherical biological aggregates represented by granular sludge, which can be composed of various substances and plays a crucial role in the formation and stability of granular sludge. Granule inner core separation technology is essential for a deep understanding of the biological and engineering characteristics of aerobic and anaerobic granular sludge, and helps to promote the progress and sustainable development of wastewater treatment technology. SUMMARY

[0009] To overcome the shortcomings of existing technologies and address the difficulty in extracting and separating the core components of spherical bioaggregates, this invention provides a method for extracting the core of spherical bioaggregates. The method uses a core separation device to extract samples, providing a foundation for subsequent microbial community analysis and structural deconstruction. By employing capillary glass tubes with different inner diameters and a needle to immobilize granular sludge, the core of spherical bioaggregates with a particle size greater than 1000 μm can be effectively extracted. This ensures that the extracted core sample accurately reflects the intrinsic characteristics of the aggregate core. Extracting the core of spherical bioaggregates for studying their structure and composition, and understanding the characteristics of the core, is also of great significance for improving wastewater treatment processes and increasing treatment efficiency.

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

[0011] A method for extracting the kernel of a spherical biological aggregate includes the following steps:

[0012] 1) Sample collection and processing

[0013] The uniformly mixed particles were removed from the homogeneous mixing stage of the microbial aggregate reactor. The particles were then taken from the sampling port of the reactor using a 50ml centrifuge tube. The mud-water mixture was then separated using a low-speed centrifuge. The supernatant and some light flocs were discharged after centrifugation. The particles were then rinsed with deionized water to complete the sample collection.

[0014] 2) Core separation

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

[0016] 3) Sample collection.

[0017] Furthermore, in step 2), the upper layer of the fixed extraction device is composed of four identical truncated square organic glass blocks arranged in opposite directions and placed on the base plate in the four directions of upper left, lower left, upper right, and lower right.

[0018] In step 2), a biological aggregate core separation device is used to separate the interior from the exterior, as follows:

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

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

[0021] 2.3) After fixing the sample, insert four capillary glass tubes with an inner diameter of 500-2000 μm into the needle along the spherical bioaggregate. Slowly push the four capillary glass tubes until the core of the particle separates from the outer phase.

[0022] In step 3), after the kernel is extracted by capillary glass tubes of different inner diameters, the extracted particle kernel is placed in the capillary glass tube. 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 a glass rotor flow meter with a range of 0.1-1 L / min through a 4 mm air tube. 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 containing 30 ml of deionized water for collection.

[0023] Furthermore, separating and extracting the core and outer layer of particles with a diameter >1000μm facilitates the analysis of the distribution of microbial community structure, thereby gaining a deeper understanding of its formation mechanism and action mechanism.

[0024] The main advantages of this invention are: it can effectively separate the outer and inner structures of particles, providing a foundation for subsequent high-throughput sequencing analysis of the core microbial community structure. This invention is simple to operate and highly accurate. Effectively understanding the characteristics of the core of biological aggregates is also of great significance for improving wastewater treatment processes and increasing treatment efficiency. Attached Figure Description

[0025] Figure 1 The diagram shows a device for separating the core of a biological aggregate. In the diagram, 3 represents a truncated square plexiglass block in the separation device, 4 represents a spherical biological aggregate, 5 represents a 0.12mm needle, and 6 represents a capillary glass tube.

[0026] Figure 2 for Figure 1 A magnified view of a spherical biological aggregate placed in the image, where 5 represents a 0.12 mm needle, 6 represents a capillary glass tube, and 7 represents the extracted kernel.

[0027] Figure 3 The diagram shows the steps for extracting the nucleus of a biological aggregate. In diagram a, 1 represents the bolt that fixes the extraction device, and 2 represents the base plate that fixes the extraction device. The next step, diagram b, shows placing the particle in the center of the extraction device. The next step, diagram c, shows inserting a needle vertically into the particle. Finally, diagram d shows extracting the nucleus using a capillary glass tube. Detailed Implementation

[0028] The present invention will now be further described with reference to the accompanying drawings.

[0029] Reference Figures 1-3A method for extracting the core of a spherical biological aggregate includes the following steps:

[0030] 1) Sample collection and processing

[0031] The uniformly mixed particles were removed from the homogeneous mixing stage of the microbial aggregate reactor. The particles were then taken from the sampling port of the reactor using a 50ml centrifuge tube. The mud-water mixture was then separated using a low-speed centrifuge. The supernatant and some light flocs were discharged after centrifugation. The particles were rinsed three times with deionized water to complete the sample collection.

[0032] 2) Core separation;

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

[0034] In step 2), the upper layer of the fixed extraction device is composed of four identical square truncated organic glass blocks arranged in opposite directions and placed on the bottom plate in the four directions of upper left, lower left, upper right, and lower right. Its dimensions are 20*25mm for the top surface, 40*50mm for the bottom surface, and 20mm for the height.

[0035] In step 2), a biological aggregate core separation device is used to separate the interior from the exterior, as follows:

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

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

[0038] 2.3) After fixing the sample, insert four capillary glass tubes with an inner diameter of 500-2000 μm into the needle along the spherical bioaggregate. Slowly push the four capillary glass tubes until the core of the particle separates from the outer phase.

[0039] 3) Sample collection: After extracting the core particles using capillary glass tubes of different inner diameters, the extracted core particles are placed in the capillary glass tubes. 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 a glass rotor flow meter with a range of 0.1-1 L / min through a 4 mm air tube. The glass rotor flow meter is adjusted to 0.1 L / min, and the capillary glass tube is connected to blow the core particles into a 50 ml glass beaker containing 30 ml of deionized water for collection.

[0040] Furthermore, separating and extracting the core and outer layer of particles with a diameter >1000μm facilitates the analysis of the distribution of microbial community structure, thereby gaining a deeper understanding of its formation mechanism and action mechanism.

[0041] This embodiment uses aerobic granular sludge as an example. The aerobic granular sludge was prepared under laboratory conditions of complete granulation and stable pollutant removal after 60 days of operation. The reactor influent had a chemical oxygen demand (CODcr) of 1000±25 mg / L, ammonia nitrogen of 24±1.0 mg / L, and total nitrogen of 27±1.5 mg / L. The wastewater used was artificially simulated with sodium acetate as the main carbon source. The aerobic granular sludge was cultured in a sequencing batch reactor (SBR) for 4 hours. The particle size of the aerobic granular sludge was approximately 2500–3000 μm.

[0042] The method for extracting the kernel of a spherical biological aggregate in this embodiment includes 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 50ml centrifuge tube. After standing for 5 minutes, the upper liquid and some light, loose sludge were poured out. 3-5 particles were taken out using a large-diameter pipette and rinsed three times with deionized water.

[0044] 2) Core Separation: The main body of the spherical bioaggregate core separation device is rectangular and divided into two layers. First, the bottom plate 2 of the extraction device is placed on a horizontal table. Then, the plane is fixed at the four corners with bolts 1. Take out 3-5 particles from the outlet of the aeration stage of the aerobic granular sludge reactor. At the same time, wash the particles three times with deionized water and measure their particle size using a wet fully automatic laser particle size analyzer. Use a large-diameter plastic pipette (5mm or more) to place the particles at the center end 4 of the particle extraction device. At the same time, insert two 0.12mm microneedles 5 vertically to fix the particles. Finally, use a capillary glass tube 6 to slowly insert it into the particle from the outside of the extraction device along the microneedles. The core 7 of the particle is inside the capillary glass tube.

[0045] 3) Sample collection: Connect one end of the core in the capillary glass tube to the gas tube, connect the oxygen pump to the glass rotor flow meter and adjust its parameters to 0.1L / min. Then, use a plastic adapter to connect the gas tube and blow the particle core of the capillary glass tube into a 50ml glass beaker containing deionized water.

[0046] The apparatus for implementing this method includes a bolt 1 for fixing the extraction device, a base plate 2 for fixing the extraction device, a truncated square plexiglass block 3 in the separation device, a spherical bio-aggregate 4, a 0.12mm needle 5, a capillary glass tube 6, and the extracted kernel 7.

[0047] The embodiments described in this specification are merely examples of implementations of the inventive concept and are for illustrative purposes only. The scope of protection of this invention should not be considered limited to the specific forms described in these embodiments; rather, it extends to equivalent technical means conceived by those skilled in the art based on the inventive concept.

Claims

1. A method for extracting a core of a granular sludge spheromorph bioaggregate, characterized by, The method comprises the following steps: 1) sample collection and processing The uniformly mixed particles are taken from the uniform mixing stage of the microbial aggregate reactor, the particles are taken from the sampling port position of the reactor using a 50 ml centrifuge tube, and then the sludge-water mixture is separated using a low-speed centrifuge; the supernatant and part of the light floc after centrifugation are discharged, and the sample collection is completed by washing the particles with deionized water; 2) core separation The main body of the spheroid-like biological aggregate core separation device adopted is rectangular and is divided into two layers, the bottom of the reactor is made of a glass plate, and the four right angles of the bottom plate of the extraction device are fixed by bolts to fix the bottom plate plane; The upper layer of the fixed extraction device is composed of four identical four-prism organic glass blocks, which are placed in the left upper, left lower, right upper and right lower positions of the bottom plate; the biological aggregate core separation device is used to separate the inside and outside, and the method is as follows: 2.1) Place the sample particles in the center of the four four-prism glass blocks; 2.2) Two 0.12 mm microneedles are inserted vertically to fix the particles; 2.3) After fixing the sample, four capillary glass tubes with an inner diameter of 500-2000 μm are inserted along the needle in turn, and the four capillary glass tubes are slowly pushed until the core and the outside of the particles are separated; 3) sample collection.

2. The method of claim 1, wherein the granular sludge spheroid bioaggregate is a granular sludge spheroid bioaggregate of claim 1. In step 3), after the core is extracted using capillary glass tubes with different inner diameters, the extracted core is in the capillary glass tube, an oxygen increasing pump with a flow rate of 35 L / min, a power of 20 W and a pressure of 0.01 Mpa is connected to a glass rotor flowmeter with a range of 0.1-1 L / min to adjust the flow rate, 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 beaker containing 30 ml of deionized water for collection.

3. The method of claim 2, wherein the granular sludge spheroid core is extracted by, After the core and the outside of the particles with a particle size of >1000 μm are separated and extracted, it is convenient to analyze the microbial community structure distribution and further understand the formation mechanism and mechanism.

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