Method for evaluating degradation performance of biodegradable plastic based on microculture

Abstract

The invention discloses a method for evaluating the degradation performance of biodegradable plastic based on microculture, and belongs to the field of biodegradable materials. The method specifically comprises the following steps: 1) pre-treating a to-be-evaluated sample, and putting the pre-treated sample into a microbody culture system containing bioplastic degrading bacteria for shake culture to obtain an intermediate degradation product; and 2) separating out micro plastic particles with the particle size of more than 1 mu m and nano plastic particles with the particle size of 0.1-1 mu m and less than 0.1 mu m. (3) identifying the micro-plastic particles by adopting high-resolution vibration spectrum imaging and calculating the number of the micro-plastic particles; a high-resolution vibration spectrum method and a nano infrared vibration spectrum method are respectively adopted to carry out spatial imaging on the multi-feature functional groups of the nano plastic particles, and the number is calculated after images are superposed. The ratio of the number of the micro-plastic particles to the number of the nano-plastic particles is used as a degradation performance evaluation parameter. According to the method, microbody culture is adopted, the culture volume is reduced, the culture time is shortened, the micro-nano plastic particles are used as evaluation indexes, the degradation performance can be rapidly evaluated, the accuracy is good, and the application prospect is wide.

Description

Technical Field

[0001] The invention belongs to the field of biodegradable materials, and in particular relates to a method for evaluating the degradation performance of biodegradable plastics based on micro-body culture. Background Art

[0002] The environmental pollution problem of traditional plastics is becoming increasingly prominent. Biodegradable plastics are mass-produced and widely used as alternative materials. In order to improve the performance and service life of biodegradable plastics, they are usually modified according to their functional requirements during the production process of plastic products, including adding different types and proportions of biomass or additives, or blending multiple biodegradable plastics in different proportions. These modification treatments will lead to differences in the degradation performance of different biodegradable plastic products and may cause environmental problems to varying degrees. Therefore, it is of great significance to evaluate the degradation performance of biodegradable plastic products.

[0003] At present, the performance of biodegradable plastics is mainly evaluated by the disintegration degree and mineralization rate of biodegradable plastics during composting, but the existing evaluation methods have the following limitations:

[0004] (1) The existing compost volume is large, and most methods use a total dry solid of more than 500g; (2) The composting or cultivation cycle is long, mostly 45 days to 6 months; (3) The lower limit of the particle size calculated by disintegration is 2 mm, and no attention has been paid to smaller plastic particles below 2 mm, especially micro-nano plastics; (4) The quality of degradation performance is mostly evaluated by calculating the amount of CO2 generated, which is easily affected by interference from other composting biomass or the operation process.

[0005] Therefore, based on the outstanding problems of low efficiency and single index in the existing degradation performance evaluation of biodegradable plastic products, it is urgent to develop a method that can efficiently, quickly and multi-indexly evaluate the degradation performance of biodegradable plastic products, so as to provide a reference for the modification of biodegradable plastic products on the market. Summary of the invention

[0006] The purpose of the present invention is to provide a method for evaluating the degradation performance of biodegradable plastics based on micro-culture in order to address the prominent problems of low efficiency and single index in the prior art of evaluating the degradation performance of biodegradable plastic products.

[0007] The specific technical solutions adopted by the present invention are as follows:

[0008] The present invention provides a method for evaluating the degradation performance of biodegradable plastics based on micro-culture, and the specific steps are as follows:

[0009] S1: After pretreatment, the biodegradable plastic sample to be evaluated is placed in a micro-culture system containing bioplastic degrading bacteria for oscillation culture to biodegrade and obtain intermediate degradation products;

[0010] S2: Separate the intermediate degradation products in the micro-culture system to obtain microplastic particles with a particle size greater than 1 μm and nanoplastic particles with a particle size in the range of 0.1 to 1 μm and a particle size less than 0.1 μm;

[0011] S3: Use high-resolution vibrational spectroscopy imaging to identify microplastic particles with a particle size greater than 1 μm at the single particle level, and calculate the number of microplastic particles in the microculture system; use high-resolution vibrational spectroscopy and nano-infrared vibrational spectroscopy to perform spatial imaging of the multi-characteristic functional groups of nanoplastic particles with a particle size in the range of 0.1 to 1 μm and a particle size less than 0.1 μm, respectively, and calculate the number of nanoplastic particles in the microculture system by superimposing the spatial imaging images;

[0012] S4: The ratio of the number of microplastic particles to the number of nanoplastic particles is used as a parameter for evaluating the degradation performance of the biodegradable plastic sample.

[0013] Preferably, the pretreatment in step S1 refers to cleaning, freeze-drying and grinding the biodegradable plastic sample to be evaluated into a particle size range of 40 to 60 μm for standby use.

[0014] Preferably, the bioplastic degrading bacteria in step S1 adopts the in-situ composting Geobacillus Parageobacillus_toebii PMBT002, the depository of which is the Guangdong Provincial Microbial Culture Collection Center of the Institute of Microbiology, Guangdong Academy of Sciences, the address of which is: 5th Floor, Building 59, No. 100, Xianlie Middle Road, Guangzhou, the deposit number is GDMCCNo: 63295, and the deposit date is March 27, 2022;

[0015] The micro-culture system includes: 0.7 g L -1 K2HPO4·3H2O, 0.7 g L -1 KH2PO4, 1.0 g L -1 NH4Cl, 0.5 g L -1 NaCl, 0.2 g L -1 MgSO4·7H2O, 0.002 g L -1 FeSO4·7H2O, 0.002gL -1 ZnSO4·7H2O and 0.001 g L -1 MnSO4·H2O.

[0016] Preferably, the ratio of the biodegradable plastic sample to the micro-culture system in step S1 is 3-8 mg / L; the OD of the bioplastic degrading bacteria solution in the micro-culture system is 600 The content of iodine is 0.6~1.0, and the added amount is 2.5%~5% (v / v).

[0017] Preferably, the shaking culture in step S1 is carried out in an air bath shaker, the temperature is set to 60°C to 70°C, and the shaking culture is carried out at 160 to 180 r / min for 20 to 24 hours.

[0018] Preferably, the volume of the micro-culture system is greater than 0.1 mL.

[0019] Preferably, in step S2, the micro-nano plastic particles in the micro-body culture system are separated by a multi-stage membrane filtration and ultrafiltration salt washing method, and the specific steps are as follows: centrifugal separation is performed using an ultrafiltration membrane with a pore size of 3 kDa, the rotation speed is 6000 rpm / min, the centrifugation time is 15 min, and the filter membrane is washed with ultrapure water to remove salt; the particles on the filter membrane are then resuspended with anhydrous ethanol to form a suspension, and then graded filtration is performed using filter membranes with pore sizes of 1 μm and 0.1 μm, respectively, and ultrasonic elution is performed into anhydrous ethanol solution to obtain suspensions containing micro-nano plastic particles with a particle size greater than 1 μm, a particle size less than 0.1 μm, and a particle size in the range of 0.1 to 1 μm, respectively.

[0020] Preferably, the biodegradable plastic is thermoplastic biodegradable plastic PBAT.

[0021] Furthermore, in step S3, high-resolution vibrational spectroscopy is used for nanoplastic particles with a particle size in the range of 0.1 to 1 μm to image the characteristic functional groups of carbon-oxygen single bonds COC, methylene -CH2, benzene ring Ph and carboxyl C=O respectively; for nanoplastic particles with a particle size less than 0.1 μm, nano-infrared vibrational spectroscopy is used to image the characteristic functional groups of the benzene ring peak.

[0022] Preferably, in step S3, Photoshop software is used to perform spatial imaging overlay, and Image J software is used to perform particle statistics and particle size analysis.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] (1) The present invention adopts a micro-culture method, which greatly reduces the culture volume, from the traditional total dry solid of more than 500g to milligram level, greatly reducing resource consumption and experimental costs; in addition, the culture process uses high-temperature liquid culture, which significantly shortens the culture cycle and greatly reduces the degradation performance evaluation time of biodegradable plastic products from several months to within 24 hours, thereby improving the evaluation efficiency and accelerating the process of product development and quality inspection;

[0025] (2) The present invention innovatively uses the micro-nano plastic particles generated during the degradation process of biodegradable plastic products as evaluation indicators, and identifies them through high-resolution vibrational spectroscopy and nano-infrared vibrational spectroscopy imaging methods. Compared with traditional evaluation methods, the accuracy is significantly improved. This method is expected to provide strong technical support and quality assurance for the development of the biodegradable plastic industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 A flow chart of the biodegradable plastic degradation performance evaluation method based on micro-culture provided by the present invention;

[0027] Figure 2 This is a vibration spectrum imaging diagram of nanoplastic particles with a particle size of 0.1 to 1 μm after 24 hours of micro-culture in the embodiment;

[0028] Figure 3 Graph showing the difference in the number of plastic particles >1 μm (a), 0.1-1 μm (b) and <0.1 μm (c) released from the two biodegradable plastics after 24 hours of microculture in the example. DETAILED DESCRIPTION

[0029] The present invention is further described and illustrated below in conjunction with the accompanying drawings and specific embodiments. The technical features of each embodiment of the present invention can be combined accordingly without conflicting with each other.

[0030] Example

[0031] like Figure 1 As shown, this embodiment adopts a biodegradable plastic degradation performance evaluation method based on micro-culture to evaluate the degradation performance of two thermoplastic biodegradable plastic (PBAT) products available on the market. The specific steps are as follows:

[0032] (1) Sample pretreatment.

[0033] According to the difference in the content of thermoplastic biodegradable plastics (PBAT), the two plastics were named PBAT80 (80% PBAT+20% PLA) and PBAT100 (100% PBAT). The two plastic products were cleaned, air-dried, and then freeze-ground to a particle size range of 40-60 μm for standby use.

[0034] (2) Microculture

[0035] The in-situ composting Geobacillus toebii PMBT002 was selected as the bioplastic degrading bacteria in this embodiment. The strain is preserved in the Guangdong Provincial Microbial Culture Collection Center of the Institute of Microbiology, Guangdong Academy of Sciences, with an address of 5th Floor, Building 59, No. 100, Xianlie Middle Road, Guangzhou, with a preservation number of GDMCC No: 63295 and a preservation date of March 27, 2022.

[0036] OD 600 =0.8 of in situ composting Bacillus spp. was inoculated into 200 mL micro-culture system at 2.5% (v / v). The micro-culture system includes: 0.7 g L -1 K2HPO4·3H2O, 0.7 g L -1 KH2PO4, 1.0gL -1 NH4Cl, 0.5 g L -1 NaCl, 0.2 g L -1 MgSO4·7H2O, 0.002 g L -1 FeSO4·7H2O, 0.002 g L -1 ZnSO4·7H2O and 0.001 g L -1 MnSO4·H2O.

[0037] 1 mg of PBAT80 and PBAT100 were added to each micro-culture system, and the micro-culture was carried out in an air bath oscillator. The temperature was set to 60° C. and the shaking culture was carried out at 160 r / min for 24 hours for biodegradation.

[0038] (3) Solution sample collection.

[0039] After 24 hours of biodegradation, 3 mL of solution was taken from each micro-culture system and centrifuged using an ultrafiltration membrane with a pore size of 3 kDa at a speed of 6000 rpm / min for 15 min. The membrane was washed with ultrapure water for at least 3 times to remove salt. The particles on the membrane were then resuspended with 3 mL of anhydrous ethanol to prepare a suspension.

[0040] (4) Gradual vacuum filtration.

[0041] The suspension in step (3) was graded and filtered using filter membranes with pore sizes of 1 μm and 0.1 μm, respectively. The filter membranes were collected and the material on the filter membranes was ultrasonically eluted into 3 mL of anhydrous ethanol solution to obtain suspensions containing microplastic particles with a particle size greater than 1 μm and suspensions containing nanoplastic particles with a particle size in the range of 0.1 to 1 μm and a particle size less than 0.1 μm, respectively. The suspensions were stored at 4°C for future use.

[0042] (5) Spectral analysis and processing.

[0043] 1) Analysis of plastic particles in suspension of microplastic particles with a particle size of >1μm: Take 2μL of the suspension and drop it onto a gold sheet. After drying, use high-resolution vibrational spectroscopy to perform single particle identification statistics and calculate the microplastic particles in the micro-body culture system. The results are as follows Figure 3 (a).

[0044] 2) Analysis of plastic particles in suspension of nanoplastic particles with a particle size of 0.1 to 1 μm: Take 2 μL of the suspension and drop it onto a gold sheet. After drying, select a scanning area and use high-resolution vibrational spectroscopy to analyze the carbon-oxygen single bond COC (1094 cm -1 )、methylene-CH2(1446cm -1 ), benzene ring Ph (1620cm -1 ) and carboxyl C=O (1720cm -1 ) characteristic functional groups were spatially imaged, and the spatial imaging images obtained were overlaid using Photoshop to count the particles with the four functional groups coexisting. The results are as follows Figure 2 Image J software was then used to perform particle statistics and particle size analysis, and the number of 0.1-1 μm nanoplastic particles in the micro-culture system was calculated. Figure 3 (b).

[0045] 3) Analysis of plastic particles in suspension of nanoplastic particles with a particle size of <0.1 μm: Take 1 mL of the suspension and dilute it 100 times. After adding dispersant sodium dodecyl sulfate (0.1 mM) to disperse it, take 4 μL and drop it on the gold sheet. After drying, use nano-infrared vibration spectroscopy to analyze the benzene ring peak (1602 cm -1 ) characteristic functional groups were imaged. Image J software was then used to perform particle statistics and particle size analysis, and the number of nanoplastic particles with a size of less than 0.1 μm in the micro-culture system was calculated. The results are shown in Figure 3 (c).

[0046] The calculation formula of the number of plastic particles is:

[0047]

[0048] Where:

[0049] N——Number of plastic particles, in pieces;

[0050] S——The area of ​​the filtrate dropped onto the gold sheet, in square microns (μm 2 );

[0051] S1——Scanning area, in square micrometers (μm 2 );

[0052] n——the number of plastic particles, in pieces;

[0053] L——total volume of solution, in milliliters (mL);

[0054] L1——test volume, in milliliters (mL);

[0055] D——dilution multiple.

[0056] (6) Comparison of the degradation performance of two biodegradable plastic products, PBAT80 and PBAT100.

[0057]

[0058] Where:

[0059] N MP ——The number of microplastic particles, in pieces;

[0060] N NP ——Number of nanoplastic particles, in pieces.

[0061] The ratio R of the number of microplastic particles to the number of nanoplastic particles is used as the degradation performance evaluation parameter of the biodegradable plastic sample. The higher the R value, the worse the degradation performance of the plastic sample. Conversely, the lower the R value, the better the degradation performance.

[0062] In this example, according to the above results, within the same time (24h), the number of microparticles (>1μm) released by PBAT80 is about 5.03×10 9 The number of released nanoparticles (0.1-1 μm and <0.1 μm) was about 1.31×10 15 The R value is 3.8×10 -3 ; The number of microparticles (>1μm) released by PBAT100 is about 3.68×10 12 The number of released nanoparticles (<1 μm) is about 1.26×10 15 The R value is 2.9×10 -3 This indicates that the degradation performance of the test sample PBAT100 obtained in this embodiment is better than that of the test sample PBAT80.

[0063] The above-described embodiment is only a preferred solution of the present invention, but it is not intended to limit the present invention. A person skilled in the relevant technical field may make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, any technical solution obtained by equivalent replacement or equivalent transformation falls within the protection scope of the present invention.

Claims

1. A method for evaluating the degradation performance of biodegradable plastics based on micro-culture, characterized in that: The specific steps are as follows: S1: After pretreatment, the biodegradable plastic sample to be evaluated is placed in a micro-culture system containing bioplastic degrading bacteria for oscillation culture to biodegrade and obtain intermediate degradation products; S2: Separate the intermediate degradation products in the micro-culture system to obtain microplastic particles with a particle size greater than 1 μm and nanoplastic particles with a particle size in the range of 0.1 to 1 μm and a particle size less than 0.1 μm; S3: Use high-resolution vibrational spectroscopy imaging to identify microplastic particles with a particle size greater than 1 μm at the single particle level, and calculate the number of microplastic particles in the microculture system; use high-resolution vibrational spectroscopy and nano-infrared vibrational spectroscopy to perform spatial imaging of the multi-characteristic functional groups of nanoplastic particles with a particle size in the range of 0.1 to 1 μm and a particle size less than 0.1 μm, respectively, and calculate the number of nanoplastic particles in the microculture system by superimposing the spatial imaging images; S4: The ratio of the number of microplastic particles to the number of nanoplastic particles is used as a parameter for evaluating the degradation performance of the biodegradable plastic sample.

2. The method for evaluating the degradation performance of biodegradable plastics based on micro-culture according to claim 1, characterized in that: The pretreatment in step S1 refers to cleaning, freeze-drying and grinding the biodegradable plastic sample to be evaluated into a particle size range of 40 to 60 μm for standby use.

3. The method for evaluating the degradation performance of biodegradable plastics based on micro-culture according to claim 1, characterized in that: The bioplastic degrading bacteria in step S1 are Parageobacillus_toebiiPMBT002, which is deposited in the Guangdong Provincial Microbial Culture Collection Center of the Institute of Microbiology, Guangdong Academy of Sciences. The address of the depository is 5th Floor, Building 59, No. 100, Xianlie Middle Road, Guangzhou. The deposit number is GDMCC No: 63295, and the deposit date is March 27, 2022; The micro-culture system includes: 0.7 g L -1 K2HPO4·3H2O, 0.7 g L -1 KH2PO4, 1.0 g L -1 NH4Cl, 0.5 g L -1 NaCl, 0.2 g L -1 MgSO4·7H2O, 0.002 g L -1 FeSO4·7H2O, 0.002gL -1 ZnSO4·7H2O and 0.001 g L - 1 MnSO4·H2O.

4. The method for evaluating the degradation performance of biodegradable plastics based on micro-culture according to claim 1, characterized in that: In step S1, the ratio of the biodegradable plastic sample to the micro-culture system is 3-8 mg / L; the OD of the bioplastic degrading bacteria solution in the micro-culture system is 600 The content of iodine is 0.6~1.0, and the added amount is 2.5%~5% (v / v).

5. The method for evaluating the degradation performance of biodegradable plastics based on micro-culture according to claim 1, characterized in that: The shaking culture in step S1 is carried out in an air bath shaker, the temperature is set to 60°C to 70°C, and the shaking culture is carried out at 160 to 180 r / min for 20 to 24 hours.

6. The method for evaluating the degradation performance of biodegradable plastics based on micro-culture according to claim 1, characterized in that: The volume of the micro-culture system is 0.1 mL or more.

7. The method for evaluating the degradation performance of biodegradable plastics based on micro-culture according to claim 1, characterized in that: In step S2, the micro-nano plastic particles in the micro-body culture system are separated by a multi-stage membrane filtration and ultrafiltration salt washing method, and the specific steps are as follows: centrifugal separation is performed using an ultrafiltration membrane with a pore size of 3 kDa, the rotation speed is 6000 rpm / min, the centrifugation time is 15 minutes, and the filter membrane is washed with ultrapure water to remove salt; the particles on the filter membrane are then resuspended with anhydrous ethanol to form a suspension, and then graded filtration is performed using filter membranes with pore sizes of 1 μm and 0.1 μm, respectively, and ultrasonic elution is performed into anhydrous ethanol solution to obtain suspensions containing micro-nano plastic particles with a particle size greater than 1 μm, a particle size less than 0.1 μm, and a particle size in the range of 0.1 to 1 μm.

8. The method for evaluating the degradation performance of biodegradable plastics based on micro-culture according to claim 1, characterized in that: The biodegradable plastic is thermoplastic biodegradable plastic PBAT.

9. The method for evaluating the degradation performance of biodegradable plastics based on micro-culture according to claim 8, characterized in that: In step S3, high-resolution vibrational spectroscopy is used for nanoplastic particles with a particle size in the range of 0.1 to 1 μm to image the characteristic functional groups of carbon-oxygen single bonds COC, methylene -CH2, benzene ring Ph and carboxyl C=O respectively; for nanoplastic particles with a particle size less than 0.1 μm, nano-infrared vibrational spectroscopy is used to image the characteristic functional groups of the benzene ring peak.

10. The method for evaluating the degradation performance of biodegradable plastics based on micro-culture according to claim 1, characterized in that: In step S3, Photoshop software is used to overlay the spatial imaging images, and Image J software is used to perform particle statistics and particle size analysis.

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