Sampling device and method for in-situ cultivation of aerobic fermentation microplastic biofilm

Abstract

The present invention relates to the technical field of microplastics, and provides a sampling device and method for in-situ cultivation of microplastic biofilms by aerobic fermentation. The sampling device for in-situ cultivation of microplastic biofilms by aerobic fermentation includes a plurality of cage units, which are stacked in sequence from bottom to top; each cage unit includes a cage body, an inner net plate, a cover body assembly and an adjusting assembly. The cage body, the inner net plate and the cover body assembly are all provided with a plurality of through holes. An opening is provided at the upper part of the cage body, and the cover body assembly is arranged at the opening. By adjusting the height of the inner net plate through the adjusting assembly, the space of the cultivation chamber can be better adjusted, and the reduction of the mixture can be observed; by filling the stacking material in the air separation chamber, the microplastics can continuously maintain good contact with the stacking material during the fermentation process, effectively improving the growth state of the simulated microplastic biofilm in the real fermentation environment. At the same time, the inner net plate, as a key device in the subsequent treatment and washing process, improves the treatment efficiency.

Description

Technical Field

[0001] The present invention relates to the technical field of microplastics, and particularly to a sampling device and method for in-situ cultivation of microplastic biofilms by aerobic fermentation. Background Art

[0002] As a new type of pollutant in the natural environment, microplastics have a size less than 5 mm. They are degraded into smaller-sized microplastics by natural processes such as physical abrasion, ultraviolet irradiation, and weathering. They are commonly present in the release and decomposition of macroplastics during agricultural waste raw materials and raw material pretreatment processes, and can be transmitted and accumulated along the food chain in the ecosystem. Currently, the widespread presence of microplastics has been found in common aerobic fermentation agricultural waste raw materials such as livestock and poultry manure and crop straw. Aerobic fermentation technology is the main way for the resource utilization of agricultural waste, with the characteristics of environmental friendliness, simplicity, and economy. Under the action of appropriate oxygen conditions, it can quickly decompose the organic matter in the raw materials and release heat to effectively and harmlessly treat the fermentation raw materials, and is an important source for producing soil organic fertilizers and cattle bedding.

[0003] Under the special environmental action of aerobic fermentation, microorganisms are easily adsorbed and colonized on the surface of microplastics to form biofilms. As carriers of pollutants, pathogenic microorganisms, and antibiotic resistance genes, biofilms can cause the accumulation of harmful substances, which may lead to the migration of microplastic pollution between different environmental media during various resource utilization processes such as fertilization and bedding laying of fermentation products, bringing potential risks to the resource utilization of fermentation products and posing serious hazards to the ecosystem and human health. Therefore, it is urgent to comprehensively carry out research on the pollution status and ecological risks of microplastics in the aerobic fermentation environment.

[0004] In the prior art, the technology for in-situ cultivation of microplastic surface biofilms in the natural environment is mainly applied in the fields of soil environment and water environment. Most of them use mesh bags made of materials such as nylon and polyethylene as cultivation devices. This mesh bag method is suitable for soil and water environments with relatively small changes in environmental conditions and media. However, in the aerobic fermentation pile, there are material characteristics and environmental conditions with drastic changes such as high temperature, high humidity, and pile volume reduction. As the fermentation progresses, the volume of the materials in the pile and the mesh bag will decrease, resulting in the formation of cavities in the mesh bag. Therefore, under the influence of the upper pile materials and the cavities in the mesh bag, the deformable mesh bag will be compressed and folded, causing a stronger extrusion effect on the microplastics, which not only affects the growth of the surface biofilm, but even destroys the formed composition distribution in the biofilm, making it impossible to effectively continuously cultivate and study the evolution of the microplastic biofilm during the fermentation process. Moreover, it will further hinder the full contact between microplastics and raw materials, is not conducive to simulating the real state of microplastic biofilms in the aerobic fermentation environment, and is difficult to judge the accuracy of research data. Summary of the Invention

[0005] The present invention provides a sampling device for in-situ cultivation of microplastic biofilms in aerobic fermentation, aiming to solve the problems in the prior art that there is poor contact between microplastics and raw materials and it is impossible to simulate the growth of microplastic biofilms in an aerobic fermentation environment.

[0006] The present invention provides a sampling device for in-situ cultivation of microplastic biofilms in aerobic fermentation, comprising:

[0007] A plurality of cage units, which are stacked in sequence from bottom to top; each cage unit includes a cage body, an inner mesh plate, a cover body assembly and an adjustment assembly. The cage body, the inner mesh plate and the cover body assembly are all provided with a plurality of through holes. An opening is provided at the upper part of the cage body, and the cover body assembly is arranged at the opening; the inner mesh plate is arranged inside the cage body and divides the inside of the cage body into an air separation chamber above the inner mesh plate and a cultivation chamber below the inner mesh plate; the adjustment assembly is connected to the inner mesh plate and the cover body assembly, and the adjustment assembly is used to adjust the height of the inner mesh plate to change the volumes of the air separation chamber and the cultivation chamber.

[0008] According to the sampling device for in-situ cultivation of microplastic biofilms in aerobic fermentation provided by the present invention, the aperture of the through hole is less than 5 mm. The cover body assembly includes a cover plate and a cover body support frame, and the cover plate is arranged at the bottom of the cover body support frame; a sampling port is arranged on the side wall of the cage body, a door body connected to the side wall of the cage body is arranged at the sampling port, and a scale line is arranged on one side of the sampling port.

[0009] According to the sampling device for in-situ cultivation of microplastic biofilms in aerobic fermentation provided by the present invention, the cover body support frame includes a connecting ring and a plurality of connecting rods. The plurality of connecting rods are arranged at intervals on the outer periphery of the connecting ring. The first end of the connecting rod is connected to the connecting ring, and the second end of the connecting rod extends radially.

[0010] According to the sampling device for in-situ cultivation of microplastic biofilms in aerobic fermentation provided by the present invention, the adjustment assembly includes a metal wire and a plurality of connecting pieces. The plurality of connecting pieces are arranged on the connecting rod at intervals along the length direction of the connecting rod; the first end of the metal wire is connected to the inner mesh plate, and the second end of the metal wire is connected to different connecting pieces to adjust the height of the inner mesh plate.

[0011] According to the sampling device for in-situ cultivation of microplastic biofilms in aerobic fermentation provided by the present invention, the adjustment assembly includes an adjustment rod and a snap ring. The lower end of the adjustment rod is connected to the inner mesh plate, a plurality of card slots arranged at intervals in the up-and-down direction are arranged at the upper end of the adjustment rod, the upper end of the adjustment rod penetrates through the cover plate, and the snap ring is in snap-fit connection with the card slot of the adjustment rod.

[0012] According to a sampling device for in-situ cultivation of microplastic biofilm by aerobic fermentation provided by the present invention, the cage unit further includes a buckle assembly, the buckle assembly includes a lock body and a lock catch, one of the lock body and the lock catch is arranged on the upper part of the outer side wall of the cage, and the other of the lock body and the lock catch is arranged on the lower part of the outer side wall of the cage.

[0013] According to a sampling device for in-situ cultivation of microplastic biofilm by aerobic fermentation provided by the present invention, the cage unit further includes a lock catch assembly, the lock catch assembly includes a lock core and a lock tongue, one of the lock core and the lock tongue is arranged on the upper part of the outer side wall of the cage, and the other of the lock core and the lock tongue is arranged on the edge of the cover body assembly.

[0014] According to a sampling device for in-situ cultivation of microplastic biofilm by aerobic fermentation provided by the present invention, the cage unit further includes a positioning marker, a connecting rope and a sealing bag, the positioning marker is arranged in the sealing bag, one end of the connecting rope is connected to the cover body assembly, and the other end of the connecting rope is connected to the sealing bag.

[0015] The present invention also provides a sampling method for in-situ cultivation of microplastic biofilm by aerobic fermentation, the method is based on the sampling device for in-situ cultivation of microplastic biofilm by aerobic fermentation described in any one of the above, and includes the following steps:

[0016] Take multiple portions of different types of microplastics with the same mass and particle size, and mix them with the initial material respectively according to a predetermined ratio;

[0017] Put equal amounts of the multiple microplastic mixtures into the culture bins of different cage units, put the inner mesh plate into the cage and adjust the inner mesh plate to an appropriate height, and load the initial material into the air separation bin; install the cover body assembly on the opening and fix it with the lock catch assembly; connect the sealing bag containing the positioning marker to the cover body assembly through the connecting rope to mark each cage unit;

[0018] Stack multiple cage units in sequence from bottom to top, and connect adjacent two cage units through the buckle assembly;

[0019] Put the sampling device into the central position of the aerobic fermentation pile body, and keep the positioning marker on the surface of the pile body;

[0020] Sampling is performed in different fermentation cycles. When sampling, the cage unit to be sampled is taken out according to the positioning mark, the cover assembly and the inner mesh plate of the cage unit are removed, and the quantitative sample is taken out using a sterile sampler, and placed in clean glassware or metal containers respectively; then the inner mesh plate is placed in the cage, and the height of the inner mesh plate is adjusted according to the volume of the remaining mixture in the culture bin; the pile fermentation material is loaded into the air compartment; the cover assembly is installed on the opening and fixed with a locking assembly; a plurality of the cage units are stacked in sequence from bottom to top, and two adjacent cage units are connected by a buckle assembly; the sampling device is placed in the original culture position;

[0021] The microplastics and fermentation materials in the mixture are separated to obtain a microplastic biofilm.

[0022] According to a method for sampling microplastic biofilms cultured in situ by aerobic fermentation provided by the present invention, the step of separating microplastics and fermentation materials in the mixture to obtain microplastic biofilms comprises:

[0023] The sample is loaded into a multi-stage sieve device and placed on a vibrating screen to separate and enrich the sample step by step to obtain target microplastic particles; the bottom sieve uses the cage unit as a collector to collect the target microplastic particles;

[0024] Adjust the inner mesh of the cage unit to an appropriate height to ensure that the inner mesh does not squeeze the target microplastic particles; install the cover assembly on the opening and fix it with a lock assembly; vertically and slowly place the cage unit into a non-plastic container filled with sterile water so that the liquid level is higher than the inner mesh, ensure that all the target microplastic particles are immersed in the sterile water, lift the cage unit after a short immersion, replace the sterile water and immerse it multiple times to remove surface impurities of the microplastics;

[0025] The microplastics after multiple immersions are transferred to a clean centrifuge tube to obtain biofilm-loaded microplastics, which can be stored at low temperatures for subsequent observation of the biofilm surface morphology;

[0026] Adding sterilized water into the centrifuge tube, and performing oscillation and ultrasonic treatment;

[0027] The supernatant in the centrifuge tube was filtered through a 0.22 μm sterile filter membrane to obtain a biofilm of microplastics for subsequent sequencing and component analysis.

[0028] The sampling device for aerobic fermentation in situ culture of microplastic biofilm provided by the present invention can better adjust the space of the culture bin and observe the reduction of the mixture by adjusting the height of the inner mesh plate through the adjustment component; by filling the bulk material in the airtight bin, the microplastics can continue to maintain good contact with the bulk material during the fermentation process, effectively improving the growth state of the simulated microplastic biofilm in a real fermentation environment; at the same time, the inner mesh plate serves as a key device in the subsequent treatment and leaching process, thereby improving the treatment efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0030] Figure 1 It is a schematic diagram of the exploded structure of a cage unit provided in one embodiment of the present invention.

[0031] Figure 2 It is a schematic diagram of the three-dimensional structure of the cage unit provided by the present invention.

[0032] Figure 3 It is a schematic diagram of the top structure of the inner mesh plate provided by the present invention.

[0033] Figure 4 It is a schematic diagram of the top structure of the cover assembly provided by the present invention.

[0034] Figure 5 It is a schematic diagram of the three-dimensional structure of the sampling device for aerobic fermentation in situ cultivation of microplastic biofilm provided by the present invention.

[0035] Figure 6 It is a schematic diagram of the exploded structure of a cage unit provided in another embodiment of the present invention.

[0036] Reference numerals:

[0037] 1. Cage unit; 2. Cage; 3. Inner mesh plate; 4. Cover assembly; 5. Adjustment assembly; 6. Culture chamber; 7. Air chamber; 8. Cover plate; 9. Connecting ring; 10. Connecting rod; 11. Lock body; 12. Lock buckle; 13. Lock core; 14. Lock tongue; 15. Metal wire; 16. Connector; 17. Positioning mark plate; 18. Connecting rope; 19. Door body; 20. Scale line. DETAILED DESCRIPTION

[0038] To make the objectives, technical solutions, and advantages of the present invention more clear, the technical solutions of the present invention will be clearly and completely described below in conjunction with the accompanying drawings of the present invention. Apparently, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the scope of protection of the present invention.

[0039] In the description of the embodiments of the present application, it should be noted that, unless otherwise clearly defined and limited, the terms "connected" and "coupled" should be understood in a broad sense. For example, it may be a fixed connection, a detachable connection, or an integral connection; it may be a mechanical connection or an electrical connection; it may be directly connected or indirectly connected through an intermediate medium. For those of ordinary skill in the art, the specific meanings of the above terms in the embodiments of the present application can be understood according to specific circumstances.

[0040] In the embodiments of the present application, unless otherwise clearly defined and limited, the first feature being "on" or "under" the second feature may be that the first and second features are in direct contact, or the first and second features are indirectly in contact through an intermediate medium. Moreover, the first feature being "above", "over", and "on top of" the second feature may be that the first feature is directly above or obliquely above the second feature, or simply means that the first feature has a higher horizontal height than the second feature. The first feature being "under", "beneath", and "underneath" the second feature may be that the first feature is directly below or obliquely below the second feature, or simply means that the first feature has a lower horizontal height than the second feature.

[0041] In the description of this specification, the descriptions with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples", etc., mean that the specific features, structures, materials, or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the embodiments of the present application. In this specification, the schematic descriptions of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described can be combined in a suitable manner in any one or more embodiments or examples. In addition, without contradiction, those skilled in the art can combine and combine the different embodiments or examples described in this specification and the features of different embodiments or examples.

[0042] The following will be combined with Figures 1 to 5 , to describe the embodiments of the present invention. It should be understood that the following description is only a schematic implementation manner of the present invention and does not constitute any limitation to the present invention.

[0043] Such as Figures 1 to 5As shown in the figure, the sampling device for in-situ cultivation of aerobic fermentation microplastic biofilms includes a plurality of cage units 1, which are stacked in sequence from bottom to top; the cage unit 1 includes a cage 2, an inner net plate 3, a cover assembly 4 and an adjustment assembly 5. The cage 2, the inner net plate 3 and the cover assembly 4 are all provided with a plurality of through holes, the aperture of the through holes is less than 5 mm, an opening is provided at the upper part of the cage 2, and the cover assembly 4 is arranged at the opening. The inner net plate 3 is arranged inside the cage 2 and divides the inside of the cage 2 into an air separation chamber 7 above the inner net plate 3 and a cultivation chamber 6 below the inner net plate 3; the adjustment assembly 5 is connected to the inner net plate 3 and the cover assembly 4, and the adjustment assembly 5 is used to adjust the height of the inner net plate 3 to change the volumes of the air separation chamber 7 and the cultivation chamber 6.

[0044] For the sampling device for in-situ cultivation of aerobic fermentation microplastic biofilms provided by the present invention, by adjusting the height of the inner net plate 3 through the adjustment assembly 5, the space of the cultivation chamber 6 can be better adjusted and the reduction of the mixture can be observed; by filling the air separation chamber 7 with heap materials, the microplastics can continuously maintain good contact with the heap materials during the fermentation process, effectively improving the growth state of the simulated microplastic biofilms in the real fermentation environment. At the same time, the inner net plate 3, as a key device in the subsequent treatment and washing process, improves the treatment efficiency.

[0045] It should be noted here that, according to the particle size of the microplastics to be cultivated, in order to effectively collect the microplastics in the aerobic fermentation heap, the aperture of the through holes on the cage unit 1 should be less than the particle size of the microplastics. To study the effects of three types of microplastics on the biofilms, the sampling device of this embodiment is provided with three cage units 1, and each cage unit 1 contains a mixture of one type of microplastics, which can be used for simultaneous cultivation of three different materials of microplastics, improving the experimental flexibility and facilitating sampling at the same time. Of course, the number of cage units 1 is not limited to this, and it is specifically determined according to experimental needs.

[0046] The sampling device for in-situ cultivation of aerobic fermentation microplastic biofilms provided by the present invention has the following advantages: 1. The height-adjustable inner net plate 3 increases the contact space for in-situ cultivation, is easy to operate, and has low production costs; 2. The extended connection design facilitates the development of microplastic cultivation experiments of different types and particle sizes, and can also be sampled multiple times without affecting the continuity of cultivation; 3. The cage unit 1 can be reused. As a collection layer sieve cage in the treatment method, it can be used in cooperation with a common sieve to screen, extract and clean the samples; it has the advantages of simple structure, diverse functions, and being easy to disassemble, assemble and carry.

[0047] In an embodiment of the present invention, as Figure 4As shown, the cover assembly 4 includes a cover plate 8 and a cover support frame. The cover plate 8 is disposed at the bottom of the cover support frame. The cover support frame is used to provide support for the cover plate 8, improving the structural strength of the cover plate 8 and enabling the cover plate 8 to carry more initial materials. In this embodiment, the cage 2, the inner mesh plate 3, and the cover plate 8 are all made of metal mesh. The cage 2, the inner mesh plate 3, and the cover plate 8 adopt through holes with 35 meshes and 425 μm, preventing microplastic particles from detaching from the cage 2, and using the through holes to increase the contact of the mixture with the solid, liquid, and gas phases of the heap body, promoting the attachment and growth of microorganisms and facilitating the formation of a simulated biofilm.

[0048] In one embodiment of the present invention, as Figure 4 shown, the cover support frame includes a connecting ring 9 and a plurality of connecting rods 10. The connecting ring 9 can be circular, elliptical, or regular polygon. The plurality of connecting rods 10 are arranged at intervals on the outer periphery of the connecting ring 9. The first end of the connecting rod 10 is connected to the connecting ring 9. Specifically, the first end of the connecting rod 10 is connected to the connecting ring 9 by welding, threading, or integrally formed. The second end of the connecting rod 10 extends radially. The plurality of connecting rods 10 are all connected to the cover plate 8. Preferably, the distance between adjacent two connecting rods 10 is equal, so that the connecting rods 10 are evenly distributed, strengthening the structural strength of different regions of the cover plate 8.

[0049] In one embodiment of the present invention, the materials of the cage 2, the inner mesh plate 3, the cover assembly 4, and the adjusting assembly 5 are all corrosion-resistant stainless steel materials.

[0050] In one embodiment of the present invention, the cage 2 is cylindrical. Preferably, the outer diameter of the cage 2 is 20 cm and the axial height is 8.5 cm. The cover plate 8 is circular, and the shape of the inner mesh plate 3 matches the cross-sectional shape of the cage 2. The centers of the cover plate 8 and the inner mesh plate 3 coincide with the central axis of the cage 2.

[0051] As Figure 6 shown, a sampling port is provided on the side wall of the cage 2. The height of the sampling port is 6 cm and the width is 8 cm. A door body 19 connected to the side wall of the cage 2 is provided at the sampling port. Specifically, one side edge of the door body 19 is hinged to the side wall of the cage 2, and the other side edge of the door body 19 is connected to the side wall of the cage 2 through a fastener to lock the door body 19. When taking a small amount of samples, only the locking piece needs to be removed, and the door body 19 is opened to take samples through the sampling port. The setting of the sampling port can reduce the impact of sampling operations on the fermentation process.

[0052] Further, a scale line 20 is provided on one side of the sampling port, and numbers are provided on one side of the scale line 20. The reduced volume can be recorded according to the scale line 20, and the reduction situation can be observed through the sampling port.

[0053] The sampling device for in-situ cultivation of aerobic fermentation microplastic biofilm provided by the present invention, according to the raw material characteristics and environmental condition characteristics of large-scale aerobic fermentation, adopts a cylindrical structure as the cultivation cage body 2. The large-capacity cultivation space inside can accommodate more microplastic mixtures. At the same time, following the principle of selecting the largest possible through holes, it can increase the contact area between the microplastic mixture and the heap outside the cage body 2, and improves the disadvantages in the traditional method that may damage the structure and composition of the surface biofilm.

[0054] In an embodiment of the present invention, as Figure 1 and Figure 2 shown, the adjusting component 5 includes a metal wire 15 and a plurality of connecting pieces 16. The plurality of connecting pieces 16 are arranged on the connecting rod 10 at intervals along the length direction of the connecting rod 10. The connecting piece 16 is perpendicular to the connecting rod 10, and the connecting piece 16 is connected to the connecting rod 10 by welding, bolt connection or integrally formed. The distance between two adjacent connecting pieces 16 can be equal or unequal. The first end of the metal wire 15 is connected to the inner net plate 3. Preferably, the first end of the metal wire 15 is connected to the center of the inner net plate 3 to ensure that the inner net plate 3 remains balanced. The second end of the metal wire 15 is connected to different connecting pieces 16 to adjust the height of the inner net plate 3. Specifically, a central hole is provided in the center of the cover plate 8. The second end of the metal wire 15 passes through the central hole and then winds around the connecting piece 16 to fix the net plate in the cage body 2; by winding the second end of the metal wire 15 around different connecting pieces 16, the height of the inner net plate 3 can be adjusted.

[0055] Furthermore, the length of the metal wire 15 should be greater than the sum of the height of the cage body 2 and the radius of the cover plate 8, so that the inner net plate 3 can move at any height inside the cage body 2 and be fixedly connected to the connecting piece 16 to realize the adjustment of the space of the cultivation chamber 6. In this embodiment, the length of the metal wire 15 is 20 cm. Of course, the length of the metal wire 15 is not limited thereto and is specifically determined according to actual needs.

[0056] In an embodiment of the present invention, the air separation chamber 7 is filled with the heap material of the same period as needed to increase the contact area between the microplastics and the heap, avoid heat loss caused by cavities, and simulate the real state of the microplastics in the heap.

[0057] It should be noted that, in order to facilitate the natural colonization of the biofilm on the microplastics, after the microplastic mixture is mixed evenly, it should be naturally scattered into the inside of the cage body 2 to prevent strong extrusion and stuffing, and prevent the microplastics from piling up together due to space limitation, thereby destroying the natural contact state between the microplastics and the fermentation raw materials. Preferably, during the process of putting in the mixture and adjusting the height of the inner net plate 3, the mixture should account for about 90% of the total volume inside the cage body 2 and should not exceed 100% of the volume of the cage body 2.

[0058] Further, the through-hole diameters of the cage 2, the cover plate 8, and the inner mesh plate 3 in each cage unit 1 can be the same or different. The through-hole diameters of the cage 2, the cover plate 8, and the inner mesh plate 3 are determined by the size of the microplastics added for cultivation. The selection of the through-hole diameter should follow the following principle: while ensuring that the added microplastic particles cannot pass through the through-holes, a larger aperture should be preferably selected to increase the contact area between the microplastic mixture and the pile materials.

[0059] In another embodiment of the present invention, the adjusting assembly 5 includes an adjusting rod (not shown) and a snap ring (not shown). The adjusting rod is vertically arranged. The lower end of the adjusting rod is connected to the inner mesh plate 3. The upper end of the adjusting rod is provided with a plurality of card slots arranged at intervals in the up-and-down direction. The upper end of the adjusting rod penetrates through the cover plate 8. The snap ring is located above the cover plate 8 and is in snap-fit connection with the card slots of the adjusting rod. During use, by making the snap ring be clamped in different card slots, the height of the inner mesh plate 3 can be adjusted. When removing the inner mesh plate 3, first remove the snap ring, then remove the cover plate 8, and then take out the inner mesh plate 3.

[0060] In one embodiment of the present invention, as Figure 5 shown, the cage unit 1 further includes a buckle assembly. The buckle assembly includes a lock body 11 and a lock catch 12. One of the lock body 11 and the lock catch 12 is arranged on the upper part of the outer side wall of the cage 2, and the other of the lock body 11 and the lock catch 12 is arranged on the lower part of the outer side wall of the cage 2. Specifically, in this embodiment, the lock body 11 is arranged on the upper part of the outer side wall of the cage 2, and the lock catch 12 is arranged on the lower part of the outer side wall of the cage 2.

[0061] Preferably, two buckle assemblies are provided for each cage unit 1, and the two buckle assemblies are symmetrically arranged. The buckle assemblies on the same side of the cage 2 are on the same straight line in the up-and-down direction. When connecting two adjacent cage units 1, the lock catch 12 of the upper cage unit 1 is connected to the lock body 11 of the lower cage unit 1, and the buckle assemblies are sequentially connected to each other from top to bottom, so as to connect all the cage units 1 together, improving the connection stability between the cage units 1. By using the easily disassembled and assembled buckle assembly for the connection of the lock catch 12, the sampling device can be quickly assembled, the cultivation amount of the mixture is increased, and the sampling scheme can be flexibly designed.

[0062] In one embodiment of the present invention, as Figure 1As shown, the cage unit 1 further includes a locking component, which includes a lock core 13 and a lock tongue 14. One of the lock core 13 and the lock tongue 14 is arranged at the upper part of the outer wall of the cage body 2, and the other of the lock core 13 and the lock tongue 14 is arranged at the edge of the cover body assembly 4. Specifically, the lock core 13 is arranged at the upper part of the outer wall of the cage body 2, the lock tongue 14 is arranged at the edge of the cover body assembly 4, and the position of the lock core 13 corresponds to the position of the lock tongue 14. Preferably, a plurality of locking components are provided, and the plurality of locking components are arranged at intervals along the circumferential direction of the cage body 2. By engaging the lock core 13 with the lock tongue 14, the cover plate 8 can be fixed from different positions, improving the overall structural stability of the cage unit 1.

[0063] The sampling device for aerobic fermentation in-situ culture of microplastic biofilm provided by the present invention can reuse the cage body 2 as a collector in the sampling method treatment. The cage unit 1 has the advantages of diversified functions, short operation time of the sampling method, and high recovery rate. It can efficiently obtain microplastic particles loaded with biofilm for real-time observation, and is suitable for studying the structural evolution mechanism of biofilm on the surface of microplastics.

[0064] In an embodiment of the present invention, as Figure 5 shown, the cage unit 1 further includes a positioning marker 17, a connecting rope 18 and a sealed bag. The positioning marker 17 is arranged in the sealed bag, and the positioning marker 17 is sealed inside through the sealed bag, which can avoid the damage to the positioning marker 17 caused by water vapor, volatile organic compounds and high temperature generated by fermentation. The length of the connecting rope 18 is 200 cm. One end of the connecting rope 18 is connected to the cover body assembly 4. Specifically, one end of the connecting rope 18 is connected to the connecting rod 10 by tying a knot. The other end of the connecting rope 18 is connected to the sealed bag. Preferably, a connecting hole is designed on the sealed bag, and the other end of the connecting rope 18 is passed through the connecting hole.

[0065] During the experiment, according to different experimental design requirements, different microplastic raw materials and fermentation raw materials are mixed in proportion, the required number of cage units 1 are selected for combination, and the appropriate through-hole aperture is selected. According to different sampling requirements, multiple samplings can be carried out in the same cage unit 1. The sampling method depends on the sampling amount. If the sampling amount is large, the cover body and the inner mesh plate are removed for sampling. If the sampling amount is small, the sampling port is used to reduce the interference to the fermentation process. After sampling, the height of the inner mesh plate 3 is appropriately adjusted, and the stack materials of the same period are naturally filled into the air separation bin 7 to prevent the reduction of the contact area between the remaining mixture and the stack after sampling, which affects the continuity of cultivation. The wire 15 and the connecting rope 18 of the present invention can be made of corrosion-resistant materials as needed, but are not limited to stainless steel wires, so as to reduce the influence of the additional introduction of plastic materials on the sample and avoid the damage of the fermentation raw materials to the connecting rope 18. The length of the connecting rope 18 should be determined according to the actual stack height and working environment of the studied stack to ensure that the positioning mark is clearly visible and is not affected by the change of the stack volume during the fermentation process.

[0066] The sampling device for in-situ cultivation of microplastic biofilms in aerobic fermentation provided by the device of the present invention. The core of this sampling device lies in preventing the materials in the culture chamber 6 from being extruded by the mesh plate and increasing the contact with the materials in the heap. Moreover, it can be extended and connected to form multiple cage units 1 for simultaneous cultivation at the same location. This device reduces the impact of multiple samplings on continuous cultivation, is conducive to simulating the evolution of biofilms in actual situations, obtaining the changes of microplastic biofilms at different time periods, and is applicable to studying the formation and evolution of biofilms of different types and sizes of microplastics in aerobic fermentation, as well as the changes of various pollutants and antibiotic resistance genes on biofilms.

[0067] By reusing the cage 2 as a collector and giving play to the adjustment function of the inner mesh plate 3, the microplastics that are easy to float during cleaning are evenly distributed and all immersed in water for cleaning under the action of the inner mesh plate 3, improving the cleaning efficiency.

[0068] During the use of the sampling device, after the mixture is put into the cage 2, extrusion can be prevented by adjusting the height of the inner mesh plate 3. The metal wire 15 passes through the central hole of the cover plate 8 and is wound and fixedly connected with the connecting piece 16, realizing the fixed connection between the cover plate 8 and the inner mesh plate 3. Subsequently, the cage 2 and the cover plate 8 are closed by a buckle. The cage units 1 are stacked in sequence and connected by a buckle assembly, enabling the stable connection of each cage unit 1. The positioning mark and the cover plate 8 are fixedly connected by a connecting rope 18. The cage combination device is placed into the research heap, making the positioning mark exposed on the surface of the heap. The cage combination device is cultivated in the research heap, and microplastics are sampled at the required sampling time. As sampling and fermentation progress, the space in the culture chamber 6 gradually decreases. At this time, the height of the inner mesh plate 3 is readjusted, and the volume of the air separation chamber 7 increases. The surrounding heap materials can be filled into the air separation chamber 7 to prevent a large number of cavities from appearing in the culture chamber 6, which is not conducive to simulating the real contact state of microplastics in the heap.

[0069] In the method of post-sampling treatment, by operating to adjust the height of the inner mesh plate 3, the height of the inner mesh plate 3 is controlled to be lower than the liquid level of the sterilized water, ensuring that the floating microplastics are restricted by the inner mesh plate 3. The cage 2 is gently lifted and lowered, making all the microplastics immersed in water, and microplastics carrying biofilms with intact morphologies can be quickly obtained.

[0070] The sampling device for in-situ cultivation of microplastic biofilms in aerobic fermentation provided by the present invention has a simple structure, is easy and convenient to operate, efficient and economical, is easy to disassemble, assemble and carry, reduces the operation difficulty of sampling and treatment for experimental personnel, has an expandable material cultivation space, and has a high separation efficiency in the sampling method, which is convenient for popularization and use.

[0071] The present invention also provides a method for sampling microplastic biofilms cultured in situ by aerobic fermentation. The method is based on the sampling device for microplastic biofilms cultured in situ by aerobic fermentation described in any one of the above embodiments, and comprises the following steps:

[0072] Step S10, taking multiple portions of different types of microplastics of the same mass and particle size, and mixing them with the initial material in a predetermined ratio;

[0073] Before starting, prepare a plurality of cage units 1 in advance. The number of cage units 1 is the same as the number of microplastics. In this embodiment, three microplastics are taken.

[0074] Step S20, placing multiple equal amounts of microplastic mixtures into the culture bins 6 of different cage units 1, placing the inner mesh panel 3 into the cage 2 and adjusting the inner mesh panel 3 to a suitable height, and placing the initial material into the airtight bin 7; installing the cover assembly 4 on the opening and fixing it with a lock assembly; connecting the sealed bag equipped with the positioning mark plate 17 to the cover assembly 4 through the connecting rope 18 to mark each cage unit 1;

[0075] When the adjustment component 5 includes a metal wire 15 and a plurality of connectors 16, the height of the inner mesh plate 3 is adjusted by winding the second end of the metal wire 15 around different connectors 16. The height of the inner mesh plate 3 is adjusted according to the volume of the microplastic mixture placed in the culture chamber 6.

[0076] Step S30, stacking a plurality of cage units 1 in sequence from bottom to top, and connecting two adjacent cage units 1 by means of a buckle assembly;

[0077] The buckle components are connected so that the cage units 1 are stably connected to prevent the cage units 1 from being separated from the cage assembly device during fermentation.

[0078] Step S40, placing the sampling device in the center of the aerobic fermentation pile, and keeping the positioning mark plate 17 on the surface of the pile;

[0079] The position of the cage assembly device is located by using the positioning mark plate 17, which can facilitate subsequent operations.

[0080] Step S50: Sampling is carried out during different fermentation cycles. When sampling, the cage unit 1 to be sampled is taken out according to the positioning marker 17. The cover assembly 4 and the inner mesh plate 3 of the cage unit 1 are removed. A quantitative sample is taken out using a sterile sampler and placed in a clean glassware or metalware respectively. Then the inner mesh plate 3 is put into the cage 2, and the height of the inner mesh plate 3 is adjusted according to the volume of the remaining mixture in the culture chamber 6. The piled fermentation materials are loaded into the air separation chamber 7. The cover assembly 4 is installed on the opening and fixed using the locking component. Multiple cage units 1 are stacked sequentially from bottom to top, and adjacent two cage units 1 are connected through the buckle component. The sampling device is placed back to the original culture position.

[0081] The different fermentation cycles can be the heating period, the high-temperature period, the cooling period or other stages.

[0082] Step S60: Separate the microplastics and the fermentation materials in the mixture to obtain the microplastic biofilm.

[0083] In one embodiment of the present invention, the step of separating the microplastics and the fermentation materials in the mixture to obtain the microplastic biofilm includes:

[0084] Step S61: Load the sample into the multi-stage sieve device and place it on a vibrating sieve to perform step-by-step separation and enrichment of the sample to obtain the target microplastic particles. The bottommost sieve uses the cage unit 1 as a collector to collect the target microplastic particles.

[0085] It should be noted here that in this embodiment, the multi-stage sieve device has four layers of sieves. Among them, the topmost sieve has the largest sieve hole size, and the topmost sieve is designed to be 4 mesh, which is used to initially screen out large-size impurities. The second layer of sieve is designed to be 8 mesh, aiming to further loosen the microplastic mixture. The sieve hole size of the third layer of sieve is further refined and designed to be 16 mesh to achieve the precise separation of microplastic particles from the rest of the fermentation raw materials.

[0086] Step S62: Adjust the inner mesh plate 3 of the cage unit 1 to an appropriate height to ensure that the inner mesh plate 3 does not squeeze the target microplastic particles. The cover assembly 4 is installed on the opening and fixed using the locking component. The cage unit 1 is vertically and slowly placed into a non-plastic container filled with sterilized water, such that the liquid level is higher than the inner mesh plate 3 to ensure that all the target microplastic particles are immersed in the sterilized water. After a short immersion, the cage unit 1 is lifted, and the sterilized water is replaced for multiple immersions to remove the surface impurities of the microplastics.

[0087] It should be noted here that the temperature of the sterilized water is 4°C. Of course, the temperature of the sterilized water is not limited to this and is specifically determined according to actual needs.

[0088] Step S63: Transfer the microplastics after multiple immersions to a clean centrifuge tube to obtain the microplastics loaded with biofilm, and store them at low temperature for subsequent observation of the surface morphology of the biofilm.

[0089] Step S64: Add sterilized water into the centrifuge tube, and perform oscillation and ultrasonic treatment;

[0090] It should be noted here that the temperature of the sterilized water added into the centrifuge tube is 4°C. Of course, the temperature of the sterilized water is not limited to this, and it is specifically determined according to actual needs.

[0091] Step S65: Filter the supernatant in the centrifuge tube with a 0.22-μm sterile filter membrane to obtain the biofilm of microplastics for subsequent sequencing and component analysis.

[0092] It should be noted that the technical solutions in the various embodiments of the present invention can be combined with each other, but the basis for the combination is that those skilled in the art can implement it; when the combination of technical solutions results in contradictions or cannot be implemented, it should be considered that such a combination of technical solutions does not exist, that is, it does not belong to the protection scope of the present invention either.

[0093] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements for some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. A sampling device for in-situ cultivation of microplastic biofilm by aerobic fermentation, characterized in that: include: A plurality of cage units (1), wherein the plurality of cage units (1) are stacked in sequence from bottom to top; the cage unit (1) comprises a cage (2), an inner mesh plate (3), a cover assembly (4) and an adjustment assembly (5); the cage (2), the inner mesh plate (3) and the cover assembly (4) are all provided with a plurality of through holes; an opening is provided at the top of the cage (2), and the cover assembly (4) is arranged at the opening; the inner mesh plate (3) is arranged in the cage (2) and divides the cage (2) into an air compartment (7) located above the inner mesh plate (3) and a culture compartment (6) located below the inner mesh plate (3); the adjustment assembly (5) is connected to the inner mesh plate (3) and the cover assembly (4), and the adjustment assembly (5) is used to adjust the height of the inner mesh plate (3) so as to change the volumes of the air compartment (7) and the culture compartment (6).

2. The sampling device for aerobic fermentation in situ cultivation of microplastic biofilm according to claim 1, characterized in that: The aperture of the through hole is less than 5 mm. The cover assembly (4) comprises a cover plate (8) and a cover support frame, wherein the cover plate (8) is arranged at the bottom of the cover support frame. A sampling port is arranged on the side wall of the cage (2), and the sampling port is provided with a door (19) connected to the side wall of the cage (2). A scale line (20) is arranged on one side of the sampling port.

3. The sampling device for aerobic fermentation in situ cultivation of microplastic biofilm according to claim 2, characterized in that: The cover body support frame comprises a connecting ring (9) and a plurality of connecting rods (10), wherein the plurality of connecting rods (10) are arranged at intervals on the outer circumference of the connecting ring (9), a first end of the connecting rod (10) is connected to the connecting ring (9), a second end of the connecting rod (10) extends radially, and the plurality of connecting rods (10) are all connected to the cover plate (8).

4. The sampling device for aerobic fermentation in situ cultivation of microplastic biofilm according to claim 3, characterized in that: The adjustment assembly (5) comprises a metal wire (15) and a plurality of connecting members (16), wherein the plurality of connecting members (16) are arranged on the connecting rod (10) at intervals along the length direction of the connecting rod (10); a first end of the metal wire (15) is connected to the inner mesh plate (3), and a second end of the metal wire (15) is connected to a different connecting member (16) to adjust the height of the inner mesh plate (3).

5. The sampling device for aerobic fermentation in situ cultivation of microplastic biofilm according to claim 3, characterized in that: The adjustment assembly (5) comprises an adjustment rod and a snap ring, the lower end of the adjustment rod being connected to the inner mesh plate (3), the upper end of the adjustment rod being provided with a plurality of snap grooves spaced apart in the up-down direction, the upper end of the adjustment rod passing through the cover plate (8), the snap ring being located above the cover plate (8), and the snap ring being snap-fitted with the snap groove of the adjustment rod.

6. The sampling device for aerobic fermentation in situ cultivation of microplastic biofilm according to claim 5, characterized in that: The cage unit (1) further comprises a buckle assembly, the buckle assembly comprising a lock body (11) and a lock buckle (12), one of the lock body (11) and the lock buckle (12) being arranged at the upper part of the outer side wall of the cage body (2), and the other of the lock body (11) and the lock buckle (12) being arranged at the lower part of the outer side wall of the cage body (2); two adjacent cage units (1) are connected via the buckle assembly.

7. The sampling device for aerobic fermentation in situ cultivation of microplastic biofilm according to claim 6, characterized in that: The cage unit (1) further comprises a lock assembly, the lock assembly comprising a lock core (13) and a lock tongue (14), one of the lock core (13) and the lock tongue (14) being arranged on the upper part of the outer side wall of the cage (2), and the other of the lock core (13) and the lock tongue (14) being arranged on the edge of the cover assembly (4).

8. The sampling device for in-situ cultivation of microplastic biofilm by aerobic fermentation according to claim 7, characterized in that: The cage unit (1) further comprises a positioning mark plate (17), a connecting rope (18) and a sealing bag, wherein the positioning mark plate (17) is arranged in the sealing bag, one end of the connecting rope (18) is connected to the cover assembly (4), and the other end of the connecting rope (18) is connected to the sealing bag.

9. A method for sampling microplastic biofilms cultured in situ by aerobic fermentation, the method being based on the sampling device for microplastic biofilms cultured in situ by aerobic fermentation according to claim 8, characterized in that: The following steps are involved: Take multiple different types of microplastics of the same mass and particle size and mix them with the initial material in a predetermined proportion; Place equal amounts of a plurality of microplastic mixtures into the culture chambers (6) of different cage units (1), place an inner mesh plate (3) into the cage (2) and adjust the inner mesh plate (3) to a suitable height, and place the initial material into the airtight chamber (7); install the cover assembly (4) on the opening and secure it with a locking assembly; connect a sealed bag equipped with a positioning mark plate (17) to the cover assembly (4) via a connecting rope (18) to mark each cage unit (1); The plurality of cage units (1) are stacked in sequence from bottom to top, and two adjacent cage units (1) are connected by a buckle assembly; Place the sampling device in the center of the aerobic fermentation pile, and keep the positioning mark plate (17) on the surface of the pile; Sampling is performed during different fermentation cycles. When sampling, the cage unit (1) to be sampled is taken out according to the positioning mark plate (17), the cover assembly (4) and the inner mesh plate (3) of the cage unit (1) are removed, and a quantitative sample is taken out using a sterile sampler and placed in a clean glass container or metal container respectively; the inner mesh plate (3) is then placed in the cage (2), and the height of the inner mesh plate (3) is adjusted according to the volume of the remaining mixture in the culture chamber (6); the pile fermentation material is loaded into the air chamber (7); the cover assembly (4) is installed on the opening and fixed with a locking assembly; a plurality of the cage units (1) are stacked in sequence from bottom to top, and two adjacent cage units (1) are connected by a buckle assembly; and the sampling device is placed in the original culture position; The microplastics and fermentation materials in the mixture are separated to obtain a microplastic biofilm.

10. The sampling method for aerobic fermentation in situ culture of microplastic biofilm according to claim 9, characterized in that: The step of separating the microplastics and the fermented material in the mixture to obtain the microplastic biofilm comprises: The sample is loaded into a multi-stage sieve device and placed on a vibrating screen to separate and enrich the sample step by step to obtain target microplastic particles; the bottom sieve uses the cage unit (1) as a collector to collect the target microplastic particles; The inner mesh plate (3) of the cage unit (1) is adjusted to an appropriate height to ensure that the inner mesh plate (3) does not squeeze the target microplastic particles; the cover assembly (4) is installed on the opening and fixed using a locking assembly; the cage unit (1) is vertically and slowly placed in a non-plastic container filled with sterile water so that the liquid level is higher than the inner mesh plate (3), ensuring that all the target microplastic particles are immersed in the sterile water, and after a short immersion, the cage unit (1) is lifted up and the sterile water is replaced for multiple immersions to remove surface impurities of the microplastics; The microplastics after multiple immersions are transferred to a clean centrifuge tube to obtain biofilm-loaded microplastics, which can be stored at low temperatures for subsequent observation of the biofilm surface morphology; Adding sterilized water into the centrifuge tube, and performing oscillation and ultrasonic treatment; The supernatant in the centrifuge tube was filtered through a 0.22 μm sterile filter membrane to obtain a biofilm of microplastics for subsequent sequencing and component analysis.

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