Preparation method of micro-plastic surface biological membrane
By employing natural microbial communities and staged sampling techniques, microplastic surface biofilms were prepared, solving the problem of simulating the natural environment in existing methods, achieving ecological representativeness and research adaptability of the biofilms, and reducing costs.
Patent Information
- Application Number
- CN202511055158.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-30
- Publication Date
- 2025-12-16
AI Technical Summary
Existing methods for preparing biofilms on microplastic surfaces rely on artificial strains, which are difficult to simulate the natural environment, are complex to operate, lack universality, and cannot dynamically monitor the biofilm formation process.
Using natural microbial communities in soil solution as inoculum, microplastic surface biofilms were prepared through shaking culture and staged sampling, combined with nylon mesh bags and sterile PBS washing, adaptable to different soil types and microplastic materials.
It enhances the ecological representativeness of biofilms, provides a reliable research model, reduces experimental costs, adapts to diverse research needs, and has high reproducibility.
Smart Images
Figure CN121136823A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of biofilm culture technology on microplastics, specifically to a method for preparing a biofilm on a microplastic surface. Background Technology
[0002] Plastic pollution is one of the most pressing environmental threats facing the world today. Larger plastic fragments released through plastic pollution can be further broken down into smaller fragments, known as microplastics (MPs), through physical, chemical, and biodegradation. Microplastics readily form biofilms on their surfaces—complex communities of microorganisms and their secretions. Microplastic biofilms not only alter the physicochemical properties of microplastics but also pose potential ecological and health risks, becoming a new focus of environmental concern.
[0003] Once microplastics enter the environment, their surfaces are rapidly covered by organic matter and microorganisms, forming biofilms. Biofilms are complex and diverse in composition, including bacteria, fungi, algae, protozoa, and their secreted extracellular polymeric substances (EPS). EPS provides protection for microorganisms and promotes the aggregation and sedimentation of microplastics, thus influencing their environmental behavior.
[0004] Research on microplastic biofilms is still in its early stages. As a novel environmental pollutant, its potential ecological and health risks cannot be ignored. Strengthening related research and developing effective prevention and control measures are of great significance for protecting the ecological environment and human health. Most existing technologies require the removal of the culture medium during sampling to separate microplastics from the environment, rather than directly sampling microplastics within the culture environment to monitor the dynamic changes in microplastic surface biofilm formation. Therefore, a method for preparing and sampling microplastic surface biofilms is needed to provide a foundation for the research and analysis of microplastic surface biofilms. Summary of the Invention
[0005] Technical problems addressed: Existing methods for preparing biofilms on microplastic surfaces rely on artificial strains, making it difficult to realistically simulate natural environments. Furthermore, these methods are complex and lack versatility. Specifically, they lack standardized procedures for utilizing natural microbial communities, cannot dynamically monitor the biofilm formation process, and are ill-suited to the research needs of different soil types and microplastic materials. This invention provides a method for preparing biofilms on microplastic surfaces, offering fundamental support for research on the environmental behavior and risks of microplastics.
[0006] Technical solution: A method for preparing a biofilm on a microplastic surface, comprising the following steps: (1) loading microplastics with a particle size of 350-500 μm into a sterile nylon mesh bag with a pore size of 108 μm and sterilizing it; (2) preparing a soil solution with a soil-to-water ratio of 1:(3-7), wherein the soil solution contains a natural microbial community, and filtering it through a 0.45 μm filter membrane after centrifugation; (3) placing the sterile mesh bag containing the microplastics into the soil solution and culturing it at 20-40℃ for 7-35 days; (4) collecting microplastic samples in stages during the culturing process and rinsing them with sterile PBS at pH 7.2-7.4 to remove unattached microorganisms.
[0007] The preparation of the soil solution in step (2) includes: (a) mixing soil and ultrapure water at a soil-to-water ratio of 1:5, stirring and shaking for 1 hour; (b) centrifuging the shaken solution and filtering the supernatant through a 0.45 μm filter membrane.
[0008] In step (3), each sterilization mesh bag is filled with 50 mg of microplastics, and 5 sterilization mesh bags are placed in each glass tube.
[0009] The collection time points in step (4) are the 7th, 14th, 21st, 28th and 35th days after culture.
[0010] The soil mentioned above is any one of black soil, red soil, or alluvial soil.
[0011] The microplastics mentioned above are pure PE or PE mulch film.
[0012] The above oscillation conditions were 200 rpm and 25°C, and the centrifugation conditions were 3220 × g for 15 minutes.
[0013] The pH value of the sterile PBS mentioned in step (4) is 7.2-7.4.
[0014] In step (3), during the shaking culture, the volume ratio of soil solution to sterilized mesh bags is 30 mL of soil solution for 5 sterilized mesh bags.
[0015] Beneficial Effects: This invention utilizes natural microbial communities in soil solution as the inoculum for biofilm formation, overcoming the limitations of traditional artificial strains (such as E. coli) and more closely reflecting the interaction between microplastics and microorganisms in the actual environment. The diversity and environmental adaptability of the natural microbial community significantly enhance the ecological representativeness of the biofilm, providing a reliable model for studying the migration, aggregation, and ecological risks of microplastics in real environments. Simultaneously, by strictly limiting the soil-to-water ratio (1:5), shaking conditions (25℃, 150 rpm), and phased sampling time points (days 7, 14, 21, 28, and 35), the experimental procedure was standardized. For example, in Example 1, the total cell concentration of pure PE microplastics in the black soil environment steadily increased over 35 days (from 81,171 to 267,371 cells / mg), with small differences between parallel experiments (RSD < 15%), verifying the high reproducibility of the method. Furthermore, the phased sampling design of this invention (covering the initial colonization, maturation, and stabilization stages) combined with sterile PBS washing technology allows for precise acquisition of biofilm characteristics at different time points. Data from the examples show that the biofilm concentration of pure PE in black soil reached its peak on day 28 (249,714 cells / mg), while PE mulch in red soil exhibited rapid early adhesion (674,990 cells / mg on day 7) followed by dynamic changes, providing systematic data support for studying the succession patterns of biofilms. Third, this invention verified the universality of the method by adjusting soil type (black soil, red soil, alluvial soil) and microplastic material (pure PE, PE mulch). For example, the biofilm concentration of PE mulch in alluvial soil remained stable over 35 days (231,657 cells / mg), while the concentration fluctuation of PE mulch in red soil was related to the soil organic matter content (Examples 4-6), indicating that the method can flexibly adapt to diverse research needs. Fourth, this invention uses a 0.45μm filter membrane to filter the soil solution, effectively removing large particulate impurities while retaining dissolved organic matter and microbial inoculum (step S103), avoiding the interference of impurities on biofilm formation in traditional methods. Furthermore, the matching design of the sterilized nylon mesh bag (108 μm pore size) and the microplastic particle size (350-500 μm) prevents microplastic leakage while ensuring sufficient contact between microorganisms and the surface; the microplastic recovery rate in the examples is >99%. Finally, this invention requires no special equipment (such as a constant temperature anaerobic incubator); only a conventional shaker and centrifuge are needed to complete the operation, significantly reducing experimental costs. The examples employ simple steps such as ultrapure water washing and ordinary glass tube culture, making it suitable for laboratory and field environments and possessing high potential for widespread application. Attached Figure Description
[0016] Figure 1 This is a flowchart of the preparation method of the microplastic surface biofilm of the present invention. Detailed Implementation
[0017] The present invention will now be described in more detail with reference to specific embodiments, so as to better demonstrate the advantages of the present invention.
[0018] A method for preparing a biofilm on the surface of microplastics in water involves placing microplastics in a sterilized mesh bag into a soil solution prepared at a soil-to-water ratio of 1:5. The solution is then placed on a shaker and cultured at 150 rpm and 25°C for 14 days. Microplastic samples are collected at 7, 14, 21, 28, and 35 days. The collected microplastic samples are washed with sterile PBS to obtain microplastics that form a biofilm.
[0019] Furthermore, the sterilization mesh bags used are nylon mesh bags with a pore size of 108μm.
[0020] Note: The purpose of this step is to facilitate the recovery of microplastics that form biofilms on the surface after the biofilm cultivation step. The microplastics selected in the experiment have a particle size of 350μm-500μm, which is much larger than the 108μm pore size of the nylon mesh bag, thus preventing the microplastics from leaking out of the mesh bag. At the same time, the sterilization treatment of the nylon mesh bag has almost no effect on the formation of biofilms on the surface of the microplastics.
[0021] Furthermore, each mesh bag was filled with 50mg of microplastics and rinsed three times with ultrapure water.
[0022] Note: The purpose of this step is to eliminate any contaminants or residues that may be present on the surface of the microplastics, ensuring the accuracy of the experimental results. If there are too many microplastics in the sterilized nylon mesh bag, the microplastics will clump together due to the small size of the nylon mesh bag, which is not conducive to the formation of cell membranes on the surface of the microplastics during the subsequent shaking culture process; rinse with ultrapure water to remove surface impurities.
[0023] Furthermore, the method for preparing the soil solution used is as follows:
[0024] S101, 1:5 soil-to-water ratio, wherein the water is ultrapure water, and the mixture is thoroughly stirred with a glass rod;
[0025] S102. The mixed solution is shaken at 200 rpm at 25°C for 1 hour on a fixed-track shaker.
[0026] S103. Centrifuge the shaken solution at 3220×g for 15 minutes, and filter the supernatant through a 0.45μm filter membrane into a brown bottle.
[0027] Explanation: The purpose of this step is to simulate aquatic conditions in the natural environment, utilizing the natural microbial community in the soil as an inoculum for biofilm formation. A 1:5 soil-to-water ratio is an optimized ratio verified through experiments, balancing the concentration of microorganisms and the release of nutrients in the soil. Excessively high soil concentration may lead to an overly turbid solution, affecting microbial attachment and biofilm formation; while excessively low soil concentration may result in insufficient microbial numbers, making it difficult to form a stable biofilm. Studies have shown that a 1:5 soil-to-water ratio provides sufficient microbial inoculum and nutrients while maintaining solution transparency, which is beneficial for biofilm formation and observation. Furthermore, the 0.45μm filter membrane aims to remove large particulate impurities and some bacteria from the soil solution, while retaining dissolved organic matter and most microorganisms. The 0.45μm pore size effectively filters out particulate matter in the soil, preventing it from interfering with the formation of biofilm on the microplastic surface. In addition, the 0.45μm filter membrane can remove some bacteria but retain a sufficient number of microorganisms as an inoculum for biofilm formation. Studies have shown that soil solutions filtered through a 0.45 μm membrane can provide abundant microbial communities and nutrients, which is conducive to biofilm formation.
[0028] Furthermore, the biofilm culture method is as follows:
[0029] S201. Place the soil solution in a glass tube, and then put 5 sterile mesh bags filled with microplastics into it;
[0030] S202. Tie the glass tube to the shaker with a rubber band and set the conditions to 150 rpm and 25°C for shaking culture for 14 days.
[0031] Explanation: The purpose of this step is to simulate water flow conditions through oscillation incubation, promoting microbial attachment and biofilm formation on the microplastic surface. An oscillation frequency of 150 rpm and an incubation temperature of 25°C are used to simulate the flow and temperature conditions in natural water bodies. The 150 rpm oscillation frequency provides moderate water flow shear force, promoting microbial attachment and biofilm formation on the microplastic surface while avoiding excessive shear force that could cause biofilm detachment. The 25°C incubation temperature is close to the temperature of natural water bodies, which is conducive to microbial growth and biofilm formation. Studies have shown that an oscillation frequency of 150 rpm and an incubation temperature of 25°C effectively promote biofilm formation on the microplastic surface and closely resemble natural environmental conditions.
[0032] Furthermore, microplastic samples were collected at 7, 14, 21, 28, and 35 days, and washed with sterile PBS to obtain microplastics that formed biofilms.
[0033] Note: The purpose of this step is to study the formation process and characteristics of biofilms at different time points. Sterile PBS (phosphate-buffered saline) has a pH of 7.2-7.4, which is close to the pH of natural water bodies. This effectively removes unattached microorganisms and impurities from the surface of microplastics while avoiding damage to the biofilm. The buffering capacity of PBS maintains a stable pH, preventing pH changes from affecting the structure and function of the biofilm. Studies have shown that rinsing microplastic samples with sterile PBS effectively removes unattached microorganisms and impurities while preserving the integrity and activity of the biofilm.
[0034] Example 1
[0035] A method for preparing a microplastic surface biofilm, such as Figure 1 As shown, it includes the following steps:
[0036] S1. Sterilize the nylon mesh bags:
[0037] S101. Rinse 15 nylon mesh bags with 108μm pore size three times with ultrapure water;
[0038] S2. Sterilize the microplastics in pure PE:
[0039] S201. Fill each sterilized nylon mesh bag with 50mg of pure PE microplastics.
[0040] S202. Rinse the sterilized nylon mesh bag containing microplastics three times with ultrapure water.
[0041] S3. Prepare a black soil solution to simulate the formation of microplastic biofilms on the surface of black soil:
[0042] S301. Add 750mL of ultrapure water to 150g of black soil and stir thoroughly with a glass rod.
[0043] S302. The mixed solution is shaken at 200 rpm at 25°C for 1 hour on a fixed-track shaker.
[0044] S303. Centrifuge the shaken solution at 3220×g for 15 minutes, and filter the supernatant through a 0.45μm filter membrane into a brown bottle;
[0045] S4. The method for culturing microplastic surface biofilms in glass tubes is as follows:
[0046] S401. Place the soil filtrate obtained in the previous step into a glass tube, and then put 5 sterile mesh bags filled with microplastics into it;
[0047] S402. Add 30 mL of soil filtrate to each glass tube;
[0048] S403. Tie the glass tube to the shaker with a rubber band and set the conditions to 150 rpm and 25°C for shaking culture for 14 days.
[0049] The biofilm on the microplastic surface was sampled, and the total cell concentration was measured. The preparation method of the microplastic surface biofilm in Example 1 was used, and the total cell concentration was determined by flow cytometry (FCM). The operation steps are as follows:
[0050] 1) Microplastics were immersed in 10 mL of sterile PBS buffer and then sonicated to isolate bacteria associated with the particles. The settings used were: amplitude: 302 μm; cycle time: 30 s; pulse level: 50%; power: 50%.
[0051] 2) Biomass and planktonic bacterial concentration of the biofilm were determined by flow cytometry every two days. 1 mL of sample was stained with 10 μL of SYBR Green I (10000× dilution, Invitrogen).
[0052] 3) Flow cytometry analysis was performed using the BD Accuri C6 Plus instrument;
[0053] 4) After thorough mixing with a vortex, incubate in the dark at 37°C for 10 minutes, and select FITC-PerCP tunneling on the emission fluorescence signal excited by a blue laser at 488nm (flow rate: 66μL / min, green fluorescence tunnel: 533nm, red fluorescence tunnel: >670nm);
[0054] 5) After processing the data using BD Accuri C6 Plus software, the total cell concentration (TCC) can be measured, and the results are as follows:
[0055] The total concentration of cells on the surface of microplastics is shown in Table 1 below:
[0056] Table 1 Total cell concentration on the surface of microplastics
[0057]
[0058] As shown in Table 1 above, the method for preparing biofilms on microplastic surfaces using the present invention can effectively obtain pure PE microplastics with biofilms forming on their surfaces. Meanwhile, the significant difference in total cell concentration on the microplastic surface between 7 and 21 days indicates that the microbial community on the microplastic surface proliferates rapidly during this period.
[0059] To verify the difference between the preparation method of the microplastic surface biofilm of the present invention and the common preparation methods on the market, we conducted subsequent experiments using the total cell concentration as an indicator parameter.
[0060] Example 2
[0061] The difference between this embodiment and Embodiment 1 is that in step S301, 750mL of ultrapure water is added to 150g of red soil and the mixture is thoroughly stirred with a glass rod.
[0062] Example 3
[0063] The difference between this embodiment and Embodiment 1 is that in step S301, 750mL of ultrapure water is added to 150g of wet soil and the mixture is stirred thoroughly with a glass rod.
[0064] To verify the difference between the microplastic surface biofilm preparation method of this invention and commonly used methods, we conducted subsequent experiments using total cell concentration as an indicator parameter. The total cell concentration of microplastics forming biofilms on the surface in soil filtrate environments prepared from red soil and alluvial soil is shown in Table 2 below:
[0065] Table 2 Total cell concentration on the surface of microplastics
[0066]
[0067] As can be seen from the comparison in Table 2, although the trend of total cell concentration on the surface of pure PE microplastics in each parallel treatment of Examples 2 and 3 changed over time compared with Example 1 after changing the environmental medium, the trend was the same between the parallel treatments, and the total cell concentration was not significantly different. This may be due to the different microbial biomass in different soil environments or the different physicochemical properties of different soils. Therefore, the method for preparing biofilm on the surface of microplastics in this invention is applicable to different soil environments.
[0068] Example 4
[0069] The difference between this embodiment and Embodiment 1 is that, in step S201, 50mg of PE microplastic mulch film is placed in each sterilized nylon mesh bag.
[0070] To verify the preparation method of the microplastic surface biofilm of the present invention compared with common preparation methods on the market, we conducted subsequent experiments using the total cell concentration as an indicator parameter. The total cell concentration of the PE microplastic mulch film cultured in the black soil environment that formed a biofilm on the surface is shown in Table 3 below:
[0071] Table 3 Total cell concentration on the surface of microplastics
[0072]
[0073] As can be seen from the comparison in Table 3 above, the technical solution of this invention can effectively form a biofilm on the surface of microplastics. Furthermore, the trend of total cell concentration changes on the microplastic surface over 7-35 days indicates that this method can track the dynamic changes of the biofilm on the microplastic surface.
[0074] Example 5
[0075] The difference between this embodiment and embodiment 4 is that in step S301, 750mL of ultrapure water is added to 150g of red soil and the mixture is stirred thoroughly with a glass rod.
[0076] Example 6
[0077] The difference between this embodiment and embodiment 4 is that in step S301, 750mL of ultrapure water is added to 150g of wet soil and the mixture is stirred thoroughly with a glass rod.
[0078] To verify the preparation method of the microplastic surface biofilm of the present invention compared with common preparation methods on the market, we conducted subsequent experiments using the total cell concentration as an indicator parameter. The total cell concentration of the microplastics that formed biofilms on the surface in soil solutions prepared from red soil and alluvial soil is shown in Table 4 below:
[0079] Table 4 Total cell concentration on the surface of microplastics
[0080]
[0081] As can be seen from the comparison in Table 4 above, although the trends of total cell concentration on the surface of the microplastic film in the parallel treatments of Examples 2 and 3 changed over time compared to Example 4 after changing the environmental medium, the trends were the same between the parallel treatments, and the total cell concentrations were not significantly different. This may be due to the different microbial biomass in different soil environments or the different physicochemical properties of different soils. Therefore, the method for preparing the microplastic surface biofilm of the present invention is applicable to different soil environments.
Claims
1. A method for preparing a microplastic surface biofilm, characterized by, The method comprises the following steps: (1) microplastics with a particle size of 350-500 μm are loaded into sterilized nylon mesh bags with a pore size of 108 μm, and sterilization treatment is performed; (2) soil solution is prepared according to a soil-water ratio of 1:(3-7), wherein the soil solution comprises a natural microbial community and is treated by oscillation centrifugation and then filtered with a 0.45 μm filter membrane; (3) the sterilized mesh bag loaded with the microplastics is placed in the soil solution, and oscillation culture is performed at 20-40℃ for 7-35 days; and (4) during the oscillation culture, microplastic samples are collected in stages, and the microplastics are rinsed with sterile PBS with a pH of 7.2-7.4 to remove unattached microorganisms.
2. The production method according to claim 1, characterized by, The preparation of the soil solution in step (2) comprises: (a) mixing soil and ultrapure water according to a soil-water ratio of 1:5, stirring, and then oscillating for 1 hour; and (b) centrifuging the oscillated solution, taking the supernatant, and filtering with a 0.45 μm filter membrane.
3. The preparation method according to claim 1, characterized in that, In step (3), 50 mg of microplastics are loaded into each sterilized mesh bag, and 5 sterilized mesh bags are placed in each glass tube.
4. The preparation method according to claim 1, characterized in that, In step (4), the collection time points are the 7th day, the 14th day, the 21st day, the 28th day, and the 35th day after culture.
5. The preparation method according to claim 1, characterized in that, The soil is any one of black soil, red soil, or chao soil.
6. The method of claim 1, wherein, The microplastics are polyethylene (PE) pure products or PE geomembranes.
7. The preparation method according to claim 2, characterized in that, The oscillation condition is 200 rpm and 25℃, and the centrifugation condition is 3220×g centrifugation for 15 minutes.
8. The method of claim 1, wherein, In step (4), the pH of the sterile PBS is 7.2-7.
4.
9. The method of claim 1, wherein, In step (3), during the oscillation culture, the volume ratio of the soil solution to the sterilized mesh bag is 30 mL of soil solution corresponding to 5 sterilized mesh bags.