Method for removing biofilm on surface of micro-plastic

Abstract

The invention belongs to the technical field of biological membrane removal, and particularly relates to a method for removing a biological membrane on the surface of micro-plastic. Comprising the following steps that micro-plastic is added into an ethanol solution with the volume concentration being 30%-90% for cracking, and the adding amount of the micro-plastic is 20 g / L; carrying out ultrasonic treatment for 10-40 minutes after cracking, cleaning to remove a biological membrane which falls off after ultrasonic treatment, and drying after cleaning to obtain the micro-plastic without the biological membrane. The biological membrane on the surface of the micro-plastic is removed to the greatest extent under the condition that the physicochemical properties of the micro-plastic are not changed.

Description

Technical Field

[0001] The present invention belongs to the technical field of biofilm removal, and in particular relates to a method for removing biofilm on the surface of microplastics. Background Art

[0002] Microplastics are plastic particles smaller than 5mm in size. As emerging pollutants, they can participate in ecosystem cycles and eventually enter various animals and even the human body, posing a threat to humans and even the ecosystem. Based on their source and degradation characteristics, microplastics can be divided into traditional microplastics and biodegradable microplastics. Traditional microplastics mainly include petroleum-based polymers such as polyethylene and polypropylene. Their high molecular weight and hydrophobicity result in a microbial degradation half-life of more than 100 years in the natural environment. Biodegradable microplastics mainly include polymers such as polylactic acid and butylene terephthalate. Most of their molecular chains contain hydrolyzable ester bonds and hydrophilic functional groups, resulting in a shorter microbial degradation half-life in the natural environment. When both types of microplastics enter the environment, a biofilm composed of microorganisms and their secreted extracellular polymers will form on their surface. A biofilm is a high-density adherent aggregate whose main components are polysaccharides and proteins. Unlike other materials, the surface properties of microplastics (such as roughness and hydrophobicity) and their own chemical composition (including the composition of microplastic polymers, additives or adsorbed pollutants, etc.) have a screening effect on bacterial groups. The biofilm attached to its surface contains specific microbial communities and has a stronger ability to adsorb pollutants. The presence of biofilms not only affects the environmental behavior of microplastics, but also causes changes in the physical and chemical properties of microplastics, including increased hydrophilicity, increased density, and changes in oxygen-containing functional groups. Therefore, removing biofilms on the surface of microplastics is an important prerequisite for conducting research on the physical and chemical properties of microplastics and analyzing their environmental behavior. At present, technologies for removing biofilms have been explored to a certain extent:

[0003] Chinese patent CN202080076761.9 discloses a method for killing bacteria within a biofilm matrix and degrading the biofilm matrix using a suspension of iron oxide nanoparticles and hydrogen peroxide; Chinese patent CN202080033854.3 discloses a method for treating an aqueous system using a liquid composition of hydrogen peroxide, ascorbic acid, and fumaric acid through agitation. The hydrogen peroxide used in both methods has a strong oxidizing ability and may cause changes in the chemical properties of microplastics.

[0004] Chinese patent CN202311659560.3 discloses a method for removing bacterial biofilms using disulfate or monosulfate as an advanced oxidation system, Fe(II) as an activator, and epigallocatechin gallate as a reinforcing agent. However, iron ions are adsorbed by microplastics, interfering with the precise characterization of their physical and chemical properties.

[0005] Chinese patent CN201680059669.5 discloses a biological cleaning kit made with trypsin, but the gel used is difficult to separate from microplastics.

[0006] Liu Xiaodong et al. stated in "Fouling and Cleaning of Membranes in Membrane Bioreactors" that cleaning with clean water has a certain cleaning effect on loose biofilms on the surface and can wash away microorganisms temporarily attached to the surface of microplastics, but the effect is relatively low.

[0007] The cleaning reagents used in the above methods are expensive and the processing steps are complicated. In addition, some methods may change the physical and chemical properties of microplastics while removing biofilm, thereby affecting the accuracy of subsequent research. For these reasons, a biofilm removal method that does not affect the physical and chemical properties of microplastics is proposed. Summary of the Invention

[0008] To solve the above problems, the present invention provides a method for removing biofilm on the surface of microplastics.

[0009] A method for removing biofilm on the surface of microplastics, comprising the following steps:

[0010] The microplastics were added to an ethanol solution with a volume concentration of 40% to 80% for cracking, wherein the amount of the microplastics added was 20 g / L;

[0011] After lysis, ultrasonic treatment is performed for 10 minutes to 40 minutes, and the biofilm detached after the ultrasonic treatment is cleaned and removed. After cleaning, the microplastics with the biofilm removed are obtained by drying.

[0012] Preferably, the microplastics include traditional microplastics and biodegradable microplastics;

[0013] The traditional microplastic is any one of polypropylene, polyvinyl chloride, polyethylene and tire wear particles;

[0014] The biodegradable microplastic is any one of polylactic acid and butylene terephthalate.

[0015] Preferably, when the microplastics are traditional microplastics, the volume concentration of the ethanol solution is 70% to 80%, and the ultrasonic time is 30 minutes to 40 minutes.

[0016] Preferably, the ultrasonic frequency is 30 Hz to 50 Hz.

[0017] Preferably, when the microplastic is biodegradable microplastic, the volume concentration of the ethanol solution is 40% to 50%, and the ultrasonic time is 10 minutes to 20 minutes.

[0018] Preferably, the ultrasonic frequency is 30 Hz to 50 Hz.

[0019] Preferably, the lysis time is 25 min to 35 min.

[0020] Preferably, deionized water is used to clean the microplastics to remove the biofilm that falls off after ultrasound.

[0021] Preferably, the drying temperature is 40° C. to 55° C., and the drying time is 24 hours to 48 hours.

[0022] Compared with the prior art, the present invention is beneficial in that:

[0023] The present invention adds microplastics to an ethanol solution with a volume concentration of 40% to 80% for cracking, and the added amount of the microplastics is 20g / L; after the cracking, ultrasonic treatment is performed for 10min to 40min, and the biofilm that falls off after the ultrasound is cleaned and removed. The lysis effect of ethanol on the biofilm is combined with the stripping effect of ultrasonic treatment to remove the biofilm on the surface to the greatest extent without changing the physical and chemical properties of the microplastics. The use of this method can reduce the influence of the biofilm on the properties of the microplastics and the interference of subsequent experiments and research, providing reliable technical support for the exploration of environmental behaviors and mechanisms related to microplastics. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This figure shows the biofilm attachment on the surface of microplastics after biofilm culture experiments on traditional and biodegradable microplastics. A is the scanning electron microscope (SEM) image, B is the biofilm amount, C is the polysaccharide content, and D is the protein content.

[0025] Figure 2 SEM images of traditional and biodegradable microplastics before and after removal of surface biofilm, where A is PP, B is PVC, C is PLA, and D is PBAT.

[0026] Figure 3 The amounts of biofilm before and after the removal of surface biofilm on traditional microplastics and biodegradable microplastics, where A is PP, B is PVC, C is PLA, and D is PBAT.

[0027] Figure 4 is the biofilm removal rate of traditional microplastics and biodegradable microplastics, where A is PP, B is PVC, C is PLA, and D is PBAT.

[0028] Figure 5 The polysaccharide content of traditional microplastics and biodegradable microplastics before and after removing the surface biofilm, where A is PP, B is PVC, C is PLA, and D is PBAT.

[0029] Figure 6The protein content of traditional microplastics and biodegradable microplastics before and after removing the surface biofilm, where A is PP, B is PVC, C is PLA, and D is PBAT.

[0030] Figure 7 These are the Fourier transform infrared (FTIR) spectra of traditional and biodegradable microplastics before and after removal of surface biofilm. A is PP ultrasound for 20 min, B is PLA ultrasound for 10 min, C is PP ultrasound for 30 min, D is PLA ultrasound for 20 min, E is PP ultrasound for 40 min, and F is PLA ultrasound for 30 min.

[0031] Figure 8 The amounts of biofilm before and after the removal of surface biofilm on traditional microplastics and biodegradable microplastics, where A is PP, B is PVC, C is PLA, and D is PBAT.

[0032] Figure 9 is the biofilm removal rate of traditional microplastics and biodegradable microplastics, where A is PP, B is PVC, C is PLA, and D is PBAT.

[0033] Figure 10 The polysaccharide content of traditional microplastics and biodegradable microplastics before and after removing the surface biofilm, where A is PP, B is PVC, C is PLA, and D is PBAT.

[0034] Figure 11 The protein content of traditional microplastics and biodegradable microplastics before and after removing the surface biofilm, where A is PP, B is PVC, C is PLA, and D is PBAT.

[0035] Figure 12 The amount of biofilm on the surface of traditional microplastics before and after being lysed by 70% and 80% ethanol solutions and then ultrasonically treated for 30 and 40 minutes, where A is PE and B is TWP.

[0036] Figure 13 is the removal rate of traditional microplastic biofilm after lysis in 70% and 80% ethanol solutions and ultrasonic treatment for 30 and 40 min, where A is PE and B is TWP.

[0037] Figure 14 The polysaccharide content before and after removal of biofilm on the surface of traditional microplastics after lysis with 70% and 80% ethanol solutions and ultrasonic treatment for 30 and 40 minutes, where A is PE and B is TWP.

[0038] Figure 15 The protein content before and after the removal of biofilm on the surface of traditional microplastics after lysis with 70% and 80% ethanol solutions and ultrasonic treatment for 30 and 40 minutes, where A is PE and B is TWP.

[0039] Figure 16 The amount of biofilm before and after the removal of biofilm on the surface of traditional microplastic PP and biodegradable microplastic PLA using the Fenton method.

[0040] Figure 17 The Fenton method was used to remove the polysaccharide content before and after the surface biofilm of traditional microplastics PP and biodegradable microplastics PLA.

[0041] Figure 18 The protein content before and after the removal of biofilm on the surface of traditional microplastic PP and biodegradable microplastic PLA was determined by Fenton method.

[0042] Figure 19 Photos of microplastics after the Fenton method was used to remove biofilms from the surfaces of traditional microplastics PP and biodegradable microplastics PLA.

[0043] Figure 20 SEM images before and after the removal of biofilms on the surfaces of traditional microplastics PP and biodegradable microplastics PLA using the Fenton method.

[0044] Figure 21 These are the FTIR spectra before and after the Fenton method was used to remove the biofilm on the surface of traditional microplastics and biodegradable microplastics, where A is PP and B is PLA. DETAILED DESCRIPTION

[0045] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited by the specific embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention. The experimental methods described in the embodiments of the present invention are all conventional methods unless otherwise specified.

[0046] Table 1 Comparison table of names used in the present invention

[0047] Chinese name abbreviation Polypropylene PP polyvinyl chloride PVC Polyethylene PE Tire wear particles TWP polylactic acid PLA Butylene terephthalate PBAT Extracellular polymers EPS

[0048] Example 1

[0049] A method for removing biofilm on the surface of microplastics, comprising the following steps:

[0050] PP was added to an ethanol solution with a volume concentration of 80% and cracked for 30 minutes. The amount of microplastic added was 20 g / L. After cracking, ultrasonic treatment was performed for 40 minutes, and the biofilm detached after the ultrasound was cleaned and removed. After cleaning, the microplastic with the biofilm removed was obtained by drying.

[0051] Example 2

[0052] A method for removing biofilm on the surface of microplastics, comprising the following steps:

[0053] PVC was added to an ethanol solution with a volume concentration of 70% and cracked for 30 minutes. The amount of microplastic added was 20 g / L. After cracking, ultrasonic treatment was performed for 30 minutes, and the biofilm detached after the ultrasound was cleaned and removed. After cleaning, the microplastic with the biofilm removed was obtained by drying.

[0054] Example 3

[0055] A method for removing biofilm on the surface of microplastics, comprising the following steps:

[0056] PLA was added to a 40% volume concentration ethanol solution and cracked for 30 minutes. The amount of microplastics added was 20 g / L. After cracking, ultrasonic treatment was performed for 10 minutes, and the biofilm detached after the ultrasound was cleaned and removed. After cleaning, the microplastics with the biofilm removed were obtained by drying.

[0057] Example 4

[0058] A method for removing biofilm on the surface of microplastics, comprising the following steps:

[0059] PBAT was added to an ethanol solution with a volume concentration of 50% and cracked for 30 minutes. The amount of microplastic added was 20 g / L. After cracking, ultrasonic treatment was performed for 20 minutes, and the biofilm detached after the ultrasound was cleaned and removed. After cleaning, the microplastic with the biofilm removed was obtained by drying.

[0060] Comparative Example 1

[0061] The difference between Comparative Example 1 and Example 1 is that the volume concentration of ethanol is different, as follows:

[0062] A method for removing biofilm on the surface of microplastics, comprising the following steps:

[0063] PP was added to an ethanol solution with a volume concentration of 20% and cracked for 30 minutes. The amount of microplastic added was 20 g / L. After cracking, ultrasonic treatment was performed for 40 minutes, and the biofilm detached after the ultrasound was cleaned and removed. After cleaning, the microplastics with the biofilm removed were obtained by drying.

[0064] Comparative Example 2

[0065] The difference between Comparative Example 2 and Example 2 is that the volume concentration of ethanol is different, as follows:

[0066] A method for removing biofilm on the surface of microplastics, comprising the following steps:

[0067] PVC was added to an ethanol solution with a volume concentration of 100% and cracked for 30 minutes. The amount of microplastic added was 20 g / L. After cracking, ultrasonic treatment was performed for 30 minutes, and the biofilm detached after the ultrasound was cleaned and removed. After cleaning, the microplastic with the biofilm removed was obtained by drying.

[0068] Comparative Example 3

[0069] The difference between Comparative Example 3 and Example 3 is that the volume concentration of ethanol is different, as follows:

[0070] A method for removing biofilm on the surface of microplastics, comprising the following steps:

[0071] PLA was added to a 100% volume concentration ethanol solution and cracked for 30 minutes. The amount of microplastic added was 20 g / L. After cracking, ultrasonic treatment was performed for 10 minutes, and the biofilm detached after the ultrasound was cleaned and removed. After cleaning, the microplastic was dried to obtain the biofilm-removed microplastic.

[0072] Comparative Example 4

[0073] The difference between Comparative Example 4 and Example 4 is that the volume concentration of ethanol is different, as follows:

[0074] A method for removing biofilm on the surface of microplastics, comprising the following steps:

[0075] PBAT was added to a 20% volume concentration ethanol solution and cracked for 30 minutes. The amount of microplastics added was 20 g / L. After cracking, ultrasonic treatment was performed for 20 minutes, and the biofilm detached after the ultrasound was cleaned and removed. After cleaning, the microplastics with the biofilm removed were dried to obtain.

[0076] Verification test

[0077] 1. Effects of ethanol solution concentration and ultrasonic duration on the removal of biofilm on microplastic surfaces

[0078] The present invention selects traditional microplastics PP and PVC and biodegradable microplastics PLA and PBAT.

[0079] The traditional microplastic PP and biodegradable microplastic PLA were added to surface water at 1.25 g / L respectively, and biofilm culture was carried out for up to 210 days to obtain two types of microplastics that formed biofilms, namely, biofilm microplastics of the traditional microplastic sample PP and biofilm microplastics of the biodegradable microplastic PLA, which were respectively recorded as PP biofilm microplastics and PLA biofilm microplastics.

[0080] The traditional microplastic PVC and the biodegradable microplastic PBAT were added to surface water at 1.25 g / L, and biofilm culture was carried out for 60 days to obtain two types of microplastics that formed biofilms, namely, biofilm microplastics of the traditional microplastic sample PVC and biofilm microplastics of the biodegradable microplastic PBAT, respectively, which were recorded as PVC biofilm microplastics and PBAT biofilm microplastics. Among them, the microplastics that formed biofilms were recorded as biofilm microplastics.

[0081] Depend on Figure 1 A shows that there are microorganisms attached to the surface of biofilm microplastics. Figure 1 B can obtain the biofilm biomass of biofilm microplastics. Since EPS in biofilm is mainly composed of polysaccharides and proteins, the polysaccharide content and protein content were determined. Figure 1 C and Figure 1 D indicates the presence of polysaccharides and proteins on the surface of biofilm microplastics. This indicates that mature biofilms form on the microplastics after 210 days of culture, and biofilms form on the microplastics after 60 days of culture. Microplastic samples with biofilms attached were collected using a 100μm stainless steel mesh, washed with phosphate-buffered saline (PBS), freeze-dried, and stored at -20°C.

[0082] For each type of biofilm microplastic, the following steps were followed to remove the biofilm on the microplastic surface. Two parallel experiments were set up for each group of experiments, as follows:

[0083] (1) 200 mg of PP biofilm microplastics were added to 10 mL of ethanol solution with concentrations of 0%, 20%, 60%, 70%, 80% and 100% and lysed for 30 min. Then, the samples were placed in a CNC ultrasonic cleaner and ultrasonically treated at an ultrasonic frequency of 40 Hz for 0 min, 10 min, 20 min, 30 min, 40 min and 60 min. The microplastic samples were washed with deionized water 2 to 3 times.

[0084] 200 mg of PVC biofilm microplastics were added to 10 mL of ethanol solution with concentrations of 60%, 70%, 80% and 100% for lysis for 30 minutes, then placed in a CNC ultrasonic cleaner for ultrasonic treatment for 20%, 30% and 40 minutes, and the microplastic samples were washed with deionized water 2 to 3 times.

[0085] 200 mg of PLA biofilm microplastics were added to 10 mL of ethanol solution with concentrations of 0%, 20%, 40%, 50%, 60%, 70%, 80% and 100% for lysis for 30 minutes, and then placed in a CNC ultrasonic cleaner for ultrasonic treatment for 0 minutes, 10 minutes, 20 minutes, 30 minutes, 40 minutes and 60 minutes, and the microplastic samples were washed with deionized water 2 to 3 times.

[0086] 200 mg of PBAT biofilm microplastics were added to 10 mL of ethanol solution with concentrations of 20%, 40%, 50%, 60% and 70% and cracked for 30 minutes. Then, they were placed in a CNC ultrasonic cleaner for ultrasonic treatment for 10 minutes and 20 minutes, and the microplastic samples were washed with deionized water 2 to 3 times.

[0087] (2) The biofilm microplastic sample treated in (1) was placed in a 50°C oven and dried for 36 hours to obtain treated microplastics.

[0088] The biofilm removal rate formula is as follows:

[0089]

[0090] The biofilm removal rate was analyzed by the following indicators:

[0091] (1) The surface morphology of microplastics was observed by SEM (Nano SEM 450, Thermo Fisher Scientific).

[0092] The results are as follows Figure 2 As shown, after PP biofilm microplastics and PVC biofilm microplastics were lysed with ethanol solution and ultrasonically treated, the number of microorganisms attached to the surface of both decreased, indicating that the biofilm had fallen off. After PLA biofilm microplastics and PBAT biofilm microplastics were lysed with ethanol solution and ultrasonically treated, the number of microorganisms attached to the surface of both decreased, indicating that the biofilm had fallen off. This shows that this method is effective in removing biofilms from the surfaces of traditional and biodegradable microplastics. Among them, the biofilm is a stable structure on the surface of microplastics. After long-term cultivation, there are some loose parts that can be removed in a gentle manner (such as rinsing with clean water), corresponding to an ethanol solution concentration of 0%. However, stable biofilms colonized on the surface of microplastics require the use of ethanol solutions of other concentrations combined with ultrasonic treatment to be more thoroughly removed.

[0093] (2) The amount of biofilm was determined at 595 nm using a modified crystal violet (CV) staining method with an ultraviolet-visible spectrophotometer (UV-Vis, GENESYS 50, Thermo Fisher Scientific) as a basis for determining the efficiency of biofilm removal on the surface of biofilm-bearing microplastics.

[0094] The specific steps are as follows: take 50 mg of biofilm microplastics and an equal amount of treated microplastics, add 0.3 mL of 0.1% crystal violet staining solution and let it stand for 45 minutes to fully stain the biofilm on the surface of the microplastics. Wash it with deionized water three times and dry it at room temperature. Then transfer it to a new centrifuge tube and add 5 mL of 95% ethanol to decolorize it for 10 minutes. The absorbance of the decolorized solution is measured at 595 nm using UV-Vis.

[0095] like Figure 3As shown in Figure 2, after ethanol solution lysis and ultrasonic treatment, the corresponding biofilm amount of PP biofilm microplastics and PVC biofilm microplastics decreased. The biofilm removal rate formula was used to calculate the biofilm removal rate. Figure 4 The results showed that after lysis with 60% to 100% ethanol solution and ultrasonic treatment, the biofilm removal rates of PP biofilm microplastics and PVC biofilm microplastics could reach 72% to 98% and 87% to 99%, respectively, indicating that the biofilm removal effect of traditional microplastic surfaces after this treatment was better.

[0096] After ethanol lysis and ultrasonic treatment, the amount of biofilm on both PLA and PBAT biofilm microplastics decreased. Calculated using the biofilm removal rate formula, the biofilm removal rates for PLA and PBAT biofilm microplastics reached 79% to 96% and 43% to 77% respectively after lysis and ultrasonic treatment with 20% to 70% ethanol, indicating that this treatment effectively removed biofilm from the surfaces of biodegradable microplastics.

[0097] (3) The polysaccharide content was determined by UV-Vis (GENESYS 50, Thermo Fisher Scientific) at 490 nm using a modified phenol-sulfuric acid method with glucose as the standard; the protein content was determined by UV-Vis (GENESYS 50, Thermo Fisher Scientific) at 500 nm using a modified Lowry method with bovine serum albumin (BSA) as the standard.

[0098] The specific steps are as follows: take 20 mg of biofilm microplastics and an equal amount of treated microplastics, add 2 mL of deionized water and centrifuge at a speed of 8000 rpm in a high-speed centrifuge (LC-LXH185C, Shanghai Lichen Bangxi Instrument Technology Co., Ltd.) for 15 minutes, and the resulting suspension is used to determine the polysaccharide and protein content. The determination of polysaccharide content requires taking 1 mL of suspension, adding 1 mL of 5% phenol and 5 mL of concentrated sulfuric acid in a glass tube and letting it stand for 10 minutes. Then, place the glass tube in a 30°C water bath for 20 minutes and use UV-Vis to measure the absorbance at 490 nm. The polysaccharide content index of the microplastics before and after removing the surface biofilm is obtained by calculation through a standard curve with glucose as the standard. The remaining 1 mL was used for the determination of protein content, i.e., 0.5 mL of 0.1 mol / L sodium hydroxide was added, allowed to stand for 30 min, 5 mL of sodium carbonate alkaline copper solution (1 mL of 1% copper sulfate pentahydrate mixed with 100 mL of 2% sodium carbonate and 1 mL of sodium tartrate) was added, and after 10 min, 0.5 mL of folin phenol was added and allowed to stand for 30 min. The absorbance was measured at 500 nm using UV-Vis, and the protein content index of the microplastics before and after the removal of the surface biofilm was obtained by calculation using a standard curve with BSA as the standard.

[0099] The results are as follows Figure 5 As shown in the results, the polysaccharide content of PP biofilm microplastics, PVC biofilm microplastics, PLA biofilm microplastics, and PBAT biofilm microplastics decreased after ethanol solution lysis and ultrasonic treatment, indicating that the biofilm on the microplastic surface was reduced. The polysaccharide content of PP biofilm microplastics and PVC biofilm microplastics was lower after lysis and ultrasonic treatment with 60% to 80% ethanol solution, while the polysaccharide content of PLA biofilm microplastics and PBAT biofilm microplastics was lower after lysis and ultrasonic treatment with 40% to 70% ethanol solution. This shows that the removal effect of biofilm on the microplastic surface is better after lysis and ultrasonic treatment with high concentration (60% to 80%) ethanol solution for traditional microplastics. Biodegradable microplastics can achieve the best biofilm removal effect by lysis and ultrasonic treatment with medium concentration (40% to 50%) ethanol solution.

[0100] like Figure 6 As shown, the protein content of PP biofilm microplastics, PVC biofilm microplastics, PLA biofilm microplastics, and PBAT biofilm microplastics was reduced after ethanol solution lysis and ultrasonic treatment. When the ethanol concentration was 60% to 100%, the protein content of PP biofilm microplastics and PVC biofilm microplastics after ethanol lysis and ultrasonic treatment at this concentration was low, indicating that the removal effect of biofilm on the surface of traditional microplastics after ethanol lysis and ultrasonic treatment with a higher concentration (60% to 100%) of ethanol solution was better. When the ethanol concentration was 60% to 100%, the protein content of PLA biofilm microplastics and PBAT biofilm microplastics after ethanol lysis and ultrasonic treatment was low, indicating that biodegradable microplastics can achieve good biofilm removal effect by using a lower concentration (20% to 60%) of ethanol solution for lysis and ultrasonic treatment.

[0101] (4) FTIR spectroscopy (Vetex 70, Bruker) was used to detect changes in the functional groups of microplastics (scan range 4000 cm -1 ~400cm -1 , with a resolution of 4cm -1 , select Absorption mode).

[0102] like Figure 7 As shown, the functional groups of PP biofilm microplastics remained unchanged after ethanol lysis and ultrasonic treatment for 20, 30, and 40 minutes. Similarly, the functional groups of PLA biofilm microplastics remained unchanged after ethanol lysis and ultrasonic treatment for 10, 20, and 30 minutes. This suggests that this method can remove biofilms from the surfaces of traditional and biodegradable microplastics without affecting their molecular structure.

[0103] After analyzing the above indicators of microplastic samples before and after biofilm removal, the optimal ethanol concentration for traditional microplastics was selected as 70% to 80%, and the optimal ethanol concentration for biodegradable microplastics was selected as 40% to 50%, taking into account the removal effect, economy, safety and operational feasibility.

[0104] according to Figure 2 and Figure 7 It can be determined that the surface morphology and chemical properties of traditional microplastics will not change after the surface biofilm is removed by lysis with a 70% to 80% ethanol solution, and the surface morphology and chemical properties of biodegradable microplastics will not change after the surface biofilm is removed by lysis with a 40% to 50% ethanol solution. Therefore, after the experiment, the average, maximum, and minimum microplastic particle sizes measured before and after the removal of the microplastic surface biofilm (see Table 2) were compared to explore the effect of ultrasound on the particle size of traditional and biodegradable microplastics.

[0105] The biofilm removal rate was analyzed by the following indicators:

[0106] (1) The particles in the SEM images were counted using Nano Measurer software to determine the average, maximum, and minimum particle sizes of microplastics.

[0107] Table 2 Average, maximum and minimum microplastic particle sizes of traditional microplastic PP and biodegradable microplastic PLA before and after removal of surface biofilm

[0108]

[0109]

[0110]

[0111] As shown in Table 1, after lysis with different concentrations of ethanol and ultrasonic treatment for 30 and 40 minutes, the particle size of the traditional microplastic PP remained essentially unchanged, while the particle size of the biodegradable microplastic PLA remained essentially unchanged after ultrasonic treatment for 10 and 20 minutes. This indicates that ultrasonic treatment for 30 to 40 minutes after lysis with ethanol solution and ultrasonic treatment for 10 to 20 minutes for biodegradable microplastics are suitable for removing biofilm on the microplastic surface.

[0112] (2) Use the modified CV staining method to obtain the biofilm content index of microplastics before and after the removal of surface biofilm. The specific steps are the same as those in verification experiment 1.

[0113] like Figure 8 As shown in the figure, after being lysed by different concentrations of ethanol, the corresponding biofilm amounts of PP biofilm microplastics and PVC biofilm microplastics decreased to varying degrees after being ultrasonicated. Figure 9 After being lysed by 70% to 80% ethanol solution, the biofilm removal rate of PP biofilm microplastics treated with ultrasound for 30min, 40min and 60min was relatively high, ranging from 80% to 98%. The biofilm removal rate of PVC biofilm microplastics treated with ultrasound for 30min and 40min was relatively high, ranging from 93% to 99%. This shows that the optimal ultrasonic treatment time for removing traditional microplastic biofilms after lysis by ethanol solution is 30min and 40min. After lysis by ethanol at different concentrations, the corresponding biofilm amounts of PLA biofilm microplastics and PBAT biofilm microplastics decreased to varying degrees after ultrasound. It was calculated by verifying the formula in experiment 1. Figure 9 After lysis with 40% to 50% ethanol solution, PLA biofilm microplastics had high biofilm removal rates of 84% to 96% after ultrasonic treatment for 10, 20, 30, 40, and 60 minutes. PBAT biofilm microplastics had high biofilm removal rates of 43% to 73% after ultrasonic treatment for 10 and 20 minutes. This indicates that the optimal ultrasonic treatment time for biodegradable microplastic biofilm removal after ethanol solution lysis is 10 and 20 minutes.

[0114] (3) A modified phenol-sulfuric acid method with glucose as the standard and a modified Lowry method with BSA as the standard were used to obtain polysaccharide and protein indicators of microplastics before and after the removal of surface biofilm. The specific steps were the same as those in Validation Experiment 1.

[0115] like Figure 10 As shown, after being lysed by ethanol at different concentrations, the corresponding polysaccharide content of PP biofilm microplastics and PVC biofilm microplastics decreased to varying degrees after ultrasound treatment, indicating that the biofilm on the surface of traditional microplastics was reduced. After being lysed by ethanol at different concentrations, the corresponding polysaccharide content of PLA biofilm microplastics and PBAT biofilm microplastics decreased to varying degrees after ultrasound treatment, indicating that the biofilm on the surface of biodegradable microplastics was reduced. The polysaccharide content of PP biofilm microplastics and PVC biofilm microplastics was low after 30 minutes and 40 minutes of ultrasound treatment, indicating that the optimal ultrasound treatment time for removing traditional microplastic biofilm after lysis with ethanol solution is 30 minutes and 40 minutes. The polysaccharide content of PLAPLA biofilm microplastics and PBAT biofilm microplastics was low after ultrasound treatment, indicating that the optimal ultrasound treatment time for removing biodegradable microplastic biofilm after lysis with ethanol solution is 10 minutes and 20 minutes.

[0116] like Figure 11As shown, after being lysed by different concentrations of ethanol, the corresponding protein content of PP and PVC biofilm microplastics decreased to varying degrees after ultrasonic treatment, indicating that the biofilm on the surface of traditional microplastics was reduced. After being lysed by different concentrations of ethanol, the corresponding protein content of PLA and PBAT biofilm microplastics decreased to varying degrees after ultrasonic treatment, indicating that the biofilm on the surface of biodegradable microplastics was reduced. The protein content of PP and PVC biofilm microplastics was low after 30 and 40 minutes of ultrasonic treatment, indicating that the optimal ultrasonic treatment time for removing biofilm from traditional microplastics after lysing with ethanol solution is 30 and 40 minutes. The protein content of PLA biofilm microplastics was low after 10, 20, 30, 40, and 60 minutes of ultrasonic treatment, and the protein content of PBAT biofilm microplastics was low after 10 and 20 minutes of ultrasonic treatment, indicating that the optimal ultrasonic treatment time for removing biofilm from biodegradable microplastics after lysing with ethanol solution is 10 and 20 minutes.

[0117] From this example, we can see that for microplastics after cracking with different concentrations of ethanol, the biofilm is reduced after ultrasonic treatment, but the ultrasonic time is too long, which has a certain impact on its surface morphology. Therefore, the traditional microplastics are better removed after ultrasonic treatment for 30 minutes to 40 minutes, and the biodegradable microplastics are better removed after ultrasonic treatment for 10 minutes to 20 minutes.

[0118] 3. Examples of removing biofilms from different types of microplastic surfaces

[0119] This example selected traditional microplastics PE and TWP to verify the removal effect of biofilm on the surface of microplastics after lysis with 70% and 80% ethanol solutions and ultrasonic treatment for 30min to 40min. At the same time, the removal effect of surface biofilm of other types of traditional microplastics except PP and PVC after lysis with 70% and 80% ethanol solutions and ultrasonic treatment for 30min to 40min was explored, thereby verifying the versatility of removing biofilm on the surface of traditional microplastics.

[0120] The traditional microplastic samples were cultured in surface water for 60 days. Figure 12 、 14 and 15 can be used to obtain surface-attached biofilms. The specific preparation steps are as follows: PE and TWP microplastic samples were taken and added into surface water at 1.25 g / L respectively. Microplastic samples attached to biofilms after 60 days of culture were collected using a 100 μm stainless steel sieve to obtain PE biofilm microplastics and TWP biofilm microplastics, which were washed with PBS and then freeze-dried and stored at -20°C.

[0121] For each type of biofilm microplastic, the biofilm on the microplastic surface was removed according to the following steps. Two parallel experiments were set up for the treatment of PE and TWP microplastic samples:

[0122] (1) 200 mg of PE biofilm microplastics and TWP biofilm microplastics were added to 10 mL of 70% and 80% ethanol solutions, respectively, and lysed for 30 min. The samples were then ultrasonically treated in a CNC ultrasonic cleaner for 30 min and 40 min, respectively. The microplastic samples were washed with deionized water 2 to 3 times.

[0123] (2) The biofilm microplastics treated in step 1 were placed in a 50°C oven and dried for 36 hours.

[0124] Because according to Figure 2 、 Figure 7 As shown in Table 1, the surface morphology, average, maximum and minimum microplastic particle size and chemical properties of traditional microplastic samples after being lysed by 70% to 80% ethanol solution and then ultrasonically treated for 30 min to 40 min to remove the surface biofilm will not change. Therefore, after the end of the experiment, only the biofilm amount, biofilm removal rate, polysaccharide content and protein content of the two microplastic samples before and after the removal of the microplastic surface biofilm were compared (see Figures 12 to 15 ).

[0125] The modified CV staining method was used to obtain biofilm content before and after surface biofilm removal. The specific steps were the same as those in Validation Experiment 1.

[0126] A modified phenol-sulfuric acid method using glucose as a standard and a modified Lowry method using BSA as a standard were used to obtain polysaccharide and protein indicators of microplastics before and after removal of surface biofilm. The specific steps were the same as those in Validation Experiment 1.

[0127] like Figure 12 As shown, the amount of biofilm measured by the two microplastic samples after being lysed with 70% and 80% ethanol solutions and ultrasonically treated for 30 minutes and 40 minutes, respectively, showed a decrease to varying degrees. Figure 13 The biofilm removal rates of PE and TWP were 80-94% and 59-99%, respectively, indicating that the treatment was effective in removing the surface biofilm of traditional microplastics.

[0128] like Figure 14 As shown in the figure, the polysaccharide content of the two microplastic samples measured after lysis with 70% and 80% ethanol solutions and ultrasonic treatment for 30min and 40min decreased, indicating that the concentration of biofilm on the surface of microplastics decreased after ethanol lysis and ultrasonic treatment, which also shows that this treatment has a good effect on removing surface biofilm of traditional microplastics.

[0129] like Figure 15 As shown in the figure, the protein content of the two microplastic samples measured after lysis with 70% and 80% ethanol solutions and ultrasonic treatment for 30 minutes and 40 minutes, respectively, decreased, indicating that the treatment is effective in removing biofilms on the surface of traditional microplastics.

[0130] This example shows that the use of 70% to 80% ethanol solution for lysis and ultrasonic treatment for 30 min to 40 min is suitable for microplastics such as PE and TWP, and can effectively remove the surface biofilm of traditional microplastics.

[0131] 4. Example of using Fenton method to remove biofilm on microplastic surface

[0132] In this example, traditional microplastic PP and biodegradable microplastic PLA were selected and treated with the Fenton method to remove the biofilm on the surface of the corresponding microplastics.

[0133] The conventional microplastic PP and the biodegradable microplastic PLA were added to surface water at a concentration of 1.25 g / L for biofilm cultivation. After 210 days, mature biofilms formed on the microplastic surfaces. Microplastic samples with biofilm attachment were collected using a 100 μm stainless steel mesh. The resulting PP and PLA biofilm microplastics were then washed with PBS, freeze-dried, and stored at -20°C.

[0134] For each type of biofilm microplastic, the following steps were performed to remove the biofilm on the microplastic surface. Two parallel treatment experiments were set up:

[0135] (1) 200 mg of PP biofilm microplastics and PLA biofilm microplastics were added to 4 mL of 20 g / L ferrous sulfate solution, and 10 mL of 30% hydrogen peroxide was slowly added;

[0136] (2) placing the sample treated in step 1 in a numerically controlled ultrasonic cleaner for ultrasonic treatment for 15 minutes;

[0137] (3) Place the microplastic sample treated in step 2 in a 50°C oven and dry for 36 hours.

[0138] After the experiment, the amount of biofilm, polysaccharide content and protein content before and after the removal of biofilm on the microplastic surface were compared (see Figures 16 to 18 ) to obtain the removal of biofilm. Compare the changes in microplastic surface morphology and functional groups before and after biofilm removal (see Figures 19 to 21 and Table 3 ) to demonstrate that the Fenton method has a great influence on the physicochemical properties of traditional microplastics PP and the physical properties of biodegradable microplastics PLA.

[0139] The surface morphology of biofilm microplastic samples and those treated with the Fenton method was observed using SEM. A modified CV staining method was used to determine the biofilm content of the microplastics before and after surface biofilm removal. The specific steps were the same as those in Validation Experiment 1.

[0140] A modified phenol-sulfuric acid method using glucose as a standard and a modified Lowry method using BSA as a standard were used to obtain polysaccharide and protein indicators of microplastics before and after removal of surface biofilm. The specific steps were the same as those in Validation Experiment 1.

[0141] Table 3 Average, maximum and minimum microplastic particle sizes before and after removal of biofilm from the surface of traditional microplastic PP and biodegradable microplastic PLA using the Fenton method

[0142]

[0143] like Figure 16 As shown in the figure, after treatment with the Fenton method, the amount of biofilm on PP biofilm microplastics and PLA biofilm microplastics was extremely low, indicating that the biofilm removal effect on the microplastic surface after this treatment was better.

[0144] like Figure 17 As shown in the figure, the polysaccharide content of PP biofilm microplastics and PLA biofilm microplastics decreased after being treated by the Fenton method, indicating that the biofilm on the surface of the microplastics was reduced.

[0145] like Figure 18 As shown in the figure, the protein content of PP biofilm microplastics and PLA biofilm microplastics was reduced after being treated by Fenton method.

[0146] like Figure 19 As shown in the figure, after being treated by the Fenton method, the surface of PP biofilm microplastics and PLA biofilm microplastics appeared orange-yellow, indicating that iron ions were attached to the surface of the microplastics after the treatment.

[0147] like Figure 20 As shown in the figure, after being treated by the Fenton method, depressions and holes appeared on the surface of the biodegradable microplastic PLA particles, causing obvious damage, indicating that this method seriously affects the surface morphology of microplastics.

[0148] As shown in Table 2, after Fenton treatment, the average particle size of traditional microplastics PP and biodegradable microplastics PLA decreased by 32.4% and 31.6%, respectively, indicating that the surface morphology of the microplastics was changed after treatment.

[0149] like Figure 21 As shown in the figure, after being treated by the Fenton method, the measured FTIR spectrum shows that the traditional microplastic PP has a peak at 1651 cm -1The carbonyl peak at the position has changed, indicating that the addition of hydrogen peroxide has changed the functional groups of the microplastics.

[0150] This example shows that the removal of biofilm using the Fenton method will seriously change the physicochemical properties of traditional microplastic PP and the physical properties of biodegradable microplastic PLA.

[0151] In summary, the present invention uses a 70% to 80% ethanol solution for the lysis of traditional microplastics, followed by ultrasonic treatment for 30 to 40 minutes, and a 40% to 50% ethanol solution for the lysis of biodegradable microplastics, followed by ultrasonic treatment for 10 to 20 minutes. This method effectively removes biofilm from the microplastic surface without altering the surface morphology or chemical properties of the microplastics. This method is suitable for subsequent characterization and experimentation of microplastics and has extremely important scientific significance for in-depth research on microplastics.

[0152] It should be noted that when the claims of the present invention involve numerical ranges, it should be understood that the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. In order to avoid redundancy, the present invention describes preferred embodiments.

[0153] Although the preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0154] Obviously, those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if such changes and modifications fall within the scope of the claims and their equivalents, the present invention is intended to include such changes and modifications.

Claims

1. A method for removing biofilm on the surface of microplastics, characterized in that: The following steps are involved: The microplastics were added to an ethanol solution with a volume concentration of 40% to 80% for cracking, wherein the amount of the added microplastics was 20 g / L; After lysis, ultrasonic treatment is performed for 10 minutes to 40 minutes, and the biofilm detached after the ultrasonication is cleaned and removed. After cleaning, the microplastics with the biofilm removed are obtained by drying.

2. The method according to claim 1, characterized in that The microplastics include traditional microplastics and biodegradable microplastics; The traditional microplastic is any one of polypropylene, polyvinyl chloride, polyethylene and tire wear particles; The biodegradable microplastic is any one of polylactic acid and butylene terephthalate.

3. The method according to claim 2, characterized in that When the microplastics are traditional microplastics, the volume concentration of the ethanol solution is 70% to 80%, and the ultrasonic time is 30 minutes to 40 minutes.

4. The method according to claim 3, characterized in that The ultrasonic frequency is 30Hz~50Hz.

5. The method according to claim 2, characterized in that When the microplastics are biodegradable microplastics, the volume concentration of the ethanol solution is 40% to 50%, and the ultrasonic time is 10 minutes to 20 minutes.

6. The method according to claim 5, characterized in that The ultrasonic frequency is 30Hz~50Hz.

7. The method according to claim 1, characterized in that The lysis time is 25min to 35min.

8. The method according to claim 1, characterized in that Deionized water was used to wash the microplastics during cleaning to remove the biofilm that was detached after ultrasound.

9. The method according to claim 1, characterized in that The drying temperature is 40° C. to 55° C., and the drying time is 24 hours to 48 hours.

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