Active plastic degrading bacterium screening culture method based on combination of Raman spectrum and heavy water mark

Abstract

The invention discloses a method for screening and culturing active plastic degrading bacteria based on combination of Raman spectrum and heavy water labeling, which realizes rapid screening of high-activity plastic degrading bacteria within 7 days by utilizing intestinal microorganisms under the condition of inorganic salt culture incubation with plastic as a unique carbon source. The method comprises the following steps: dissecting and homogenizing intestinal tracts of tenebrio molitor fed with plastics under a sterile condition, harvesting enteric microorganisms through iohexol density gradient centrifugation, incubating the enteric microorganisms in an inorganic salt culture solution containing heavy water and plastics to serve as a treatment group, centrifugally cleaning an incubated to-be-detected substance, and performing Raman detection, the high-activity plastic degrading bacteria can be sorted for further downstream sequencing and culture. The method is rapid, efficient, easy and convenient to operate and suitable for rapidly screening, identifying and culturing the high-activity plastic degrading bacteria from a complex natural environment, and development of plastic biodegradation and harmless treatment processes is facilitated.

Description

Technical Field

[0001] The invention relates to the technical field of microbial screening, and in particular to a screening and culturing method for active plastic-degrading bacteria based on Raman spectroscopy combined with heavy water labeling. Background Art

[0002] The traditional model of "cultivating first, then screening" is used to discover functional microorganisms from nature, which leads to inefficient input of consumables, time-consuming and labor-intensive, and can only cultivate non-target or even irrelevant microorganisms under specific environmental conditions. At present, there is still a lack of rapid and efficient technology for mining plastic-degrading functional microbial resources. Therefore, it is urgent to develop a non-destructive, rapid, efficient and convenient screening and identification method for plastic-degrading functional microorganisms in complex environments, using Raman spectroscopy combined with heavy water labeling to first screen and then culture, so as to achieve efficient sorting and cultivation of target plastic-degrading functional bacteria. Summary of the invention

[0003] The present invention aims to provide a rapid and efficient method for screening and culturing active plastic-degrading bacteria based on Raman spectroscopy combined with heavy water labeling, so as to solve the shortcomings of traditional culturing methods for screening plastic-degrading bacteria, which are time-consuming, labor-intensive and inefficient.

[0004] The present invention proposes a method for screening and culturing active plastic-degrading bacteria based on Raman spectroscopy combined with heavy water labeling, comprising the following steps:

[0005] S1. The extracted microorganisms were added to MSM liquid medium containing heavy water and PS microplastics for incubation. At the same time, intestinal microorganisms without microplastics, Escherichia coli with PS, and Staphylococcus aureus with PS were used as negative controls, and the screened PS-degrading bacteria P. aeruginosa-IUE with PS were used as positive controls. After incubation under the same conditions, Raman detection was performed;

[0006] S2. Calculate CD / (C-D+CH) of the treatment group and the control group respectively, and use three times the average value of the negative control group plus three times the standard deviation as the discrimination criterion, and set it as a threshold with statistical difference for the detection and sorting of highly active microorganisms that degrade polystyrene microplastics. A value less than or equal to 25.72 is judged as having no degradation ability for PS plastics, and a value greater than or equal to 25.72 is judged as PS highly active plastic degrading bacteria.

[0007] Preferably, the microorganisms in step S1 are from the intestines of mealworms extracted after the mealworm larvae were fed with polystyrene foam as the only food for 42 days, and the extraction method is:

[0008] S11. Pick about 10 mealworms and soak them in 75% ethanol for 5 minutes;

[0009] S12, after being filled with sterile ultrapure water for 5 min, the intestine of mealworms was dissected on a sterile wax tray and placed in a 1.5 mL sterile centrifuge tube;

[0010] S13, add 200 μL sterile PBS phosphate buffer and homogenize with a homogenizer;

[0011] S14. The formula of PBS phosphate buffer is: Na2HPO4 1.44g, KH2PO4 0.24g, NaCl 8g, KCl 0.2g, H2O 1L.

[0012] Preferably, the extracted microbial homogenate is subjected to iohexol density gradient centrifugation to collect intestinal microorganisms in the intestinal homogenate, the collected intestinal microbial cells are washed three times by centrifugation with sterile ultrapure water, and then added to the culture medium for incubation, and the centrifugation conditions are: 4°C, 6500rpm.

[0013] Preferably, the steps of collecting intestinal microorganisms by iohexol density gradient centrifugation are:

[0014] S101, adding the intestinal homogenate into a PBS solution containing 20% ​​Tween and vortexing vigorously for 30 min on a vortex shaker to separate the cells attached to the soil particles;

[0015] S102, carefully adding an equal volume of iohexol solution to the soil suspension, followed by centrifugation at 14000 g and 4°C for 90 min;

[0016] S103. Carefully aspirate the liquid layer containing microorganisms in the middle, add 10 volumes of PBS and centrifuge at 6500 rpm for 10 minutes.

[0017] Preferably, in step S1, the culture medium is an MSM liquid culture medium with 50% heavy water added, 20 mg / mL of 200 μm particle size PS plastic is added as the sole carbon source, and the incubation time is 7 days, wherein the MSM liquid culture medium formula is: NaHPO4 2.8g; KH2PO4 1.0g; (NH4)2SO4 0.5g; CaCl2·2H2O 0.001mg; H3BO3 0.03mg; FeSO4·7H2O 0.2mg; MnCl2·4H2O 0.003mg; NiCl2·6H2O 0.002mg; Na2EDTA 0.5mg; CoCl2·6H2O 0.02mg; ZnSo4·7H2O 0.01mg; Na2MoO4·2H2O 0.003mg; H2O 1L; add 1.5% agar to the above reagents to make a solid culture medium.

[0018] Preferably, in step 1, the incubated cells are collected and washed three times with sterile ultrapure water by centrifugation at 6500 rpm, 2 μL of bacterial liquid is dropped on a glass slide covered with aluminum foil and air-dried before Raman testing; single-cell Raman spectrum is collected using a LabRAM Aramis (HORIBA Jobin-Yvon) confocal microscope Raman system equipped with a 300 grooves / mm diffraction grating; a 532 nm Nd:YAG laser is used as the excitation light and a 100x air objective lens with a numerical aperture of 0.9 (Olympus, Japan) is used to observe bacteria and collect Raman signals; the acquisition time of each Raman spectrum is 25 s, and the spectrum acquisition range is 400-3200 cm -1 .

[0019] Preferably, highly active polystyrene-degrading functional microorganisms identified by Raman are laser-ejected and sorted using a single cell sorter, and the bacteria are sorted into a receiver containing 3 μL PBS. About 20-30 single cells are collected during each sorting process, and then multiple displacement amplification is performed using a genome amplification kit (REPLI-g Single Cell Kit) according to the instructions, and finally the amplified products are subjected to metagenomic sequencing analysis.

[0020] Preferably, the residual microplastics after incubation in the culture medium for 30 days are recovered, ultrasonically cleaned and dried, and then the structural changes of the microplastics after incubation are determined by scanning electron microscopy, water contact angle, gel permeation chromatography, infrared spectrometer, thermogravimetric analysis, high performance liquid chromatography-mass spectrometry and other physical and chemical characterization methods to verify the biodegradation of PS microplastics.

[0021] Preferably, the flow Raman instrument FlowRACS is used to detect, screen and sort out highly active plastic degrading bacteria. The sorted cells are cultured on LB plates for 2 days, and then the harvested cells are placed in 100mL liquid MSM culture medium with 50mg PS microplastics added, and cultured at 30°C for 30 days. The culture is then diluted and spread on MSM agar medium with PS particles as the only carbon source, and pure colonies are screened. The primer pair 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3') are used to amplify the nearly full-length 16S rRNA gene sequence to determine its species classification status. The obtained plastic degrading bacteria are placed in MSM culture medium with PS plastic as the only carbon source and incubated for 60 days, and the residual microplastics are collected. The physical and chemical characterization methods such as infrared spectroscopy, thermogravimetric analysis and gel permeation chromatography are used to determine the structural changes of the plastics and verify the degradation ability of the bacteria on plastics. The formula of LB solid culture medium is: tryptone 10g; yeast extract 5g; NaCl 10g; H2O1L; agar 15g.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] 1) The traditional culture method for screening functional microorganisms is one-sided, blind, distorted, time-consuming and labor-intensive. The isotope labeling method based on metabolic activity labeling is fast, efficient, non-destructive, specific and stable, and can target functional microorganisms with high metabolic activity in situ environments with high throughput.

[0024] 2) Compared with the stable isotope labeling method, Raman spectroscopy combined with heavy water labeling only requires the addition of heavy water to the in situ environment and incubation for a period of time. It is inexpensive and has the characteristics of simple operation, stability, and high specificity. It can truly reflect the metabolic activity and phenotype of microorganisms in the in situ environment.

[0025] 3) Suitable for single cell or large number of cell detection, not limited by sample size or differences in microplastic types.

[0026] Therefore, in summary, the method provided by the present invention overcomes the one-sidedness, blindness and distortion of traditional culture methods, can realize the technical process of "targeted screening first, then culture", has the advantages of rapid, in situ, stable and specific, and is suitable for actual environmental sites with other plastic pollution. It is a non-destructive, rapid, efficient and convenient screening and identification method for plastic-degrading functional microorganisms in complex environments, which greatly promotes the search for efficient degradation strains or synthetic functional microorganisms to reduce plastic pollution. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 1 is a schematic diagram of a method for screening highly active polystyrene-degrading functional microorganisms in some embodiments of the present invention;

[0028] Figure 2 yes Figure 1 Raman identification and screening results of S3 in the middle;

[0029] Figure 3 yes Figure 1 Raman sorting result diagram of S5 process in;

[0030] Figure 4 This is the result of microbial metagenome assembly after Raman sorting;

[0031] Figure 5 is a scanning electron micrograph of microbial colonization on the surface of microplastics before and after incubation;

[0032] Figure 6 is the contact angle change diagram of microplastics before and after incubation;

[0033] Figure 7 This is the result graph of the molecular weight change of microplastics before and after incubation;

[0034] Figure 8 It is the infrared spectrum of the functional group changes of microplastics before and after incubation;

[0035] Fig. 9 is the thermogravimetric analysis graph of microplastics before and after incubation;

[0036] Fig.10 is the HPLC-MS / MS spectrum of the degradation products in the culture medium after incubation;

[0037] Fig.11 The results of the molecular weight changes of microplastics after incubation with sorted culture bacteria;

[0038] Fig.12 Infrared spectra of changes in functional groups of microplastics before and after incubation with sorted culture bacteria;

[0039] Fig.13 Thermogravimetric analysis of microplastics before and after incubation with sorted culture bacteria;

[0040] Fig.14 This is a graph showing the Raman identification and screening results of active polyurethane plastic degrading bacteria in Example 2. DETAILED DESCRIPTION

[0041] The present invention will be further explained below in conjunction with specific embodiments.

[0042] Example 1

[0043] A method for screening and culturing active plastic-degrading bacteria based on Raman spectroscopy combined with heavy water labeling comprises the following steps:

[0044] S1. Feed mealworm larvae with polystyrene foam for 42 days to enrich PS-degrading bacteria in the intestine. Then pick about 10 mealworms and soak them in 75% ethanol for 5 minutes, then pick them into sterile ultrapure water for 5 minutes, dissect the mealworm intestine on a sterile wax tray and pick them into a 1.5mL sterile centrifuge tube. Add 200μL sterile PBS phosphate buffer and homogenize with a homogenizer. The formula of PBS phosphate buffer is: Na2HPO4 1.44g, KH2PO4 0.24g, NaCl 8g, KCl 0.2g, H2O 1L.

[0045] S2. Collect intestinal microorganisms from intestinal homogenate by iohexol density gradient centrifugation.

[0046] S3. The intestinal microorganisms of mealworms were incubated in an inorganic salt culture medium containing heavy water and PS plastic as the treatment group, the intestinal microorganisms without PS plastic, E. coli JM103 with PS plastic, and Staphylococcus aureus RN4220 with PS plastic as the negative control group, and Pseudomonas aeruginosa-IUE with PS plastic as the positive control group. The culture medium is MSM liquid culture medium, and the formula is: NaHPO4 2.8g; KH2PO4 1.0g; (NH4)2SO4 0.5g; CaCl2·2H2O 0.001mg; H3BO3 0.03mg; FeSO4·7H2O0.2mg; MnCl2·4H2O 0.003mg; NiCl2·6H2O 0.002mg; Na2EDTA 0.5mg; CoCl2·6H2O 0.02mg; ZnSo4·7H2O 0.01mg; Na2MoO4·2H2O 0.003mg; H2O 1L; adding 1.5% agar to the above reagents is a solid culture medium. The above pure bacteria are all preserved in the laboratory, and after overnight culture in LB liquid culture medium, they are centrifuged and washed for 3 minutes at 6500rpm with sterile water for a total of 3 times before use. The inoculation amount of each microorganism in the culture solution is 1-5%, and the incubation time is 7 days. The formula of LB liquid culture medium is: tryptone 10g; yeast extract 5g; NaCl 10g, H2O 1L.

[0047] S4. After the incubated object is centrifuged and washed, it is dropped onto a low Raman background substrate for Raman detection. The CD / (C-D+CH) of the treatment group and the control group are calculated respectively, and the three times average value of the negative control group ratio plus 3 times the standard deviation is used as the discrimination standard. This threshold value indicates that there is a statistically significant difference between the experimental group and the negative control group. According to the average value of the CD ratio of the negative control bacteria + 3 times the standard deviation, the dividing line of the CD value of 25.72 can be easily obtained. It is set as a threshold with statistical differences for the detection and sorting of highly active microorganisms that degrade polystyrene microplastics. High metabolic activity indicates that it has great potential to degrade polystyrene microplastics, such as Figure 2 .

[0048] S5. After the cells with CD ratios higher than the division threshold detected by Raman are sorted out by Raman ejection, the amplified products are subjected to metagenomic sequencing and assembly analysis after multiple displacement (MDA) amplification. Figure 3. Laser ejection sorting was performed on the highly active plastic-degrading bacteria identified by Raman. After gene amplification and metagenomic sequencing, 12 highly active PS-degrading bacteria were obtained, including 5 reported plastic-degrading bacteria and 7 new unreported degrading bacteria. A high-quality spliced ​​genome was obtained, which contained a variety of plastic-degrading enzymes including hydrolases, esterases, oxidases and peroxidases. The accuracy of Raman identification and sorting was proved from the genotype ( Figure 4 ). Based on the information of degradation enzymes and degradation products, the pathways of PS degradation by intestinal bacteria were mapped, including physical destruction and aging of plastics, extracellular enzymatic oxidation and depolymerization, and intracellular metabolism and mineralization of biodegradation byproducts.

[0049] S6. Physical and chemical characterization methods such as scanning electron microscopy (SEM), water contact angle (WCA), gel permeation chromatography (GPC), infrared spectrometer (FTIR), thermogravimetric analysis (TGA), and HPLC-MS analysis were used to determine the structural changes of microplastics after 30 days of incubation to verify the biodegradation of PS microplastics. Scanning electron microscopy (SEM) results showed that the surface of PS alone in the culture medium or microplastics incubated with Escherichia coli DH5α (E. coli + PS) and Staphylococcus aureus RN4220 (SA + PS) was smooth and complete. After incubation for 30 days with PS-fed T. molitor larvae intestinal microorganisms (PS + PS) and isolated strain Pseudomonas aeruginosa-IUE (PSE + PS), obvious holes and cracks could be observed on the residual PS microplastics, and microorganisms colonized on the surface of the microplastics and formed biofilms. The changes in the contact angles of water droplets and the surface of PS microplastics showed that the contact angles of the PS + PS group and the PSE + PS group were significantly lower than those of PS in the culture medium, SA + PS, and E. coli + PS groups, such as Figure 6 The GPC results showed that after 30 days of incubation of the intestinal microorganisms of T. molitor larvae fed with PS (PS+PS) and the isolated strain of Pseudomonas aeruginosa (PSE+PS), the molecular weight of PS decreased, e.g. Figure 7 The FTIR spectrum of the residual PS in the culture medium showed that the OH stretching vibration (3550 cm -1 ), CO stretching vibration (1085cm -1 ) and C=O stretching vibration (1710 cm -1 ) and other new functional groups, indicating that PS microplastics have undergone oxidative depolymerization, such as Figure 8 The thermal stability of plastics in the PS+PS and PSE+PS groups was significantly lower than that of PS microplastics at 5% mass loss, decreasing from 362.2°C to 311.0°C and 114.7°C, respectively, indicating that new degradation intermediates with different thermal properties were generated after cultivation, such as Fig. 9In addition, the biodegradation products after incubation were also detected. The HPLC-MS spectrum results showed that low molecular weight products were formed after intestinal microorganisms degraded PS microplastics, and reported PS degradation products including acetophenone, α, α-xylene, etc. were detected. Fig.10 These characterization results demonstrated the structural changes of microplastics after incubation, verified the biodegradation of PS microplastics, and proved the reliability of this method.

[0050] Flow Raman FlowRACS (Qingdao Single Cell Biotechnology Co., Ltd.) was used to detect, screen and sort out highly active plastic degrading bacteria. The sorted cells were cultured on LB plates for 2 days, and then the harvested cells were placed in 100mL liquid MSM medium supplemented with 50mg PS microplastics and cultured at 30°C for 30 days. The culture was then diluted and spread on MSM agar medium with PS particles as the only carbon source, and pure colonies were screened. The nearly full-length 16SrRNA gene sequence was amplified using primers 27F and 1492R to determine its species classification status. Finally, two strains of PS plastic degrading bacteria were obtained, namely Arthrobacter sp.-IUE (ART) and Pseudomonas stutzeri-IUE (PST). The obtained plastic-degrading bacteria were placed in MSM culture medium with PS plastic as the only carbon source and incubated for 60 days. The residual microplastics were collected and the structural changes of the plastics were determined by FTIR, TGA, GPC and other physical and chemical characterization methods to verify the degradation ability of the bacteria on plastics. The GPC results showed that after ART and PST strains were incubated in MSM culture medium with PS as the only carbon source for 60 days, the molecular weight of the residual PS plastics recovered from the culture medium decreased significantly, such as Fig.11 The FTIR spectrum of the residual PS recovered from the culture medium showed that the OH stretching vibration (3550 cm -1 ), CO stretching vibration (1085cm -1 ) and C=O stretching vibration (1710 cm -1 ) and other new functional groups, indicating that PS microplastics have undergone oxidative depolymerization, such as Fig.12 The thermal stability of residual PS recovered from the culture medium was significantly lower than that of PS microplastics at 5% mass loss, decreasing from 363.0°C to 229.2°C and 310.3°C, respectively, indicating that new degradation intermediates with different thermal properties were generated after the culture, such as Fig.13 .

[0051] In some embodiments, Figure 2 As shown, the above step S2 may specifically include:

[0052] S201: 200 μL of intestinal homogenate was homogenized by adding 5 mL of phosphate buffer and 25 μL of Tween 20 surfactant;

[0053] S202: The homogenized intestinal sample was shaken by a vortexer for 30 minutes to separate the bacteria attached to the soil particles;

[0054] S203: Then, the mud is slowly added into the iohexol solution, wherein the specific density of the iohexol solution is 1.42 g / mL, to ensure the treatment effect.

[0055] S204: Centrifuge at 7800 rpm for 90 min at 4°C.

[0056] S205: After centrifugation, a white bacterial layer appears in the middle layer. The bacterial layer is collected and washed twice with PBS phosphate buffer to remove excess dye and impurities.

[0057] Thus, according to the embodiment of the present invention, the target conditions can be set more conveniently, thereby improving the efficiency of screening.

[0058] In some embodiments, Figure 2 As shown, the above step S5 may specifically include:

[0059] S501: Highly active cells were collected into a sterile receiver containing 3 μL sterile PBS using a Raman pulse laser ejection system (PRECI SCS, HOOKE Instruments Ltd, China). About 30 single cells were collected during each sorting process.

[0060] S502: Unsorted cells were used as negative control. The sorted cells were transferred into cell lysis buffer and whole genomic DNA was amplified by multiple displacement amplification (MDA) according to the protocol of REPLI-g single cell kit (Qiagen, Germany).

[0061] S503: The MDA product was diluted 50-fold in sterile nuclease-free water and the bacterial 16S rRNA gene was amplified using primers 341F (5′-CCTACGGGNGGCWGCAG-3′) and 785R (5′-GACTACHVGGGTATCTAATCC-3′). Only when the DNA samples of sorted cells were PCR-positive and the “No sort” control group had no PCR product, the DNA would be retained for further metagenomic sequencing.

[0062] Thus, according to the embodiment of the present invention, the target conditions can be set more conveniently, thereby improving the success rate and efficiency of screening.

[0063] Example 2

[0064] The specific screening process of this embodiment is as follows:

[0065] Taking polyurethane plastic PU as an example, the above scheme is used to screen and cultivate PU plastic degrading functional bacteria.

[0066] First, intestinal microorganisms were extracted. The microorganisms were extracted from the intestines of mealworm larvae after they were fed with polyurethane (PU) foam plastics as the only food for 42 days. The extracted intestinal microbial cells were collected by iohexol density gradient centrifugation, and then incubated for 7 days in an MSM liquid culture medium containing 50% heavy water with 2 mg / mL of PU microplastics as the only carbon source. At the same time, intestinal microorganisms without microplastics, Escherichia coli with PU, and Staphylococcus aureus with PU were used as negative controls, and the screened PU-degrading bacteria P. aeruginosa-IUE with PU were used as positive controls. After incubation under the same conditions, Raman detection was performed. The CD / (C-D+CH) of the treatment group and the control group were calculated respectively, and the three times the average value of the negative control group ratio plus 3 times the standard deviation were used as the discrimination criteria. The dividing line of the CD value of 12.40 can be easily obtained, as shown in Fig.14 . This threshold indicates that there is a statistically significant difference between the experimental group and the negative control group, and can be used to detect, sort and culture highly active microorganisms that degrade polyurethane plastics. Flow Raman was used to sort out highly active PU-degrading bacteria, and then the harvested cells were placed in liquid MSM culture medium supplemented with 50 mg of PU microplastics and cultured at 30°C for 30 days. The culture was then diluted and spread on MSM agar medium with PU particles as the only carbon source, and two pure strains were obtained by screening, namely Pseudomonas aeruginosa-IUE and Stutzerimonas stutzeri-IUE. The obtained plastic-degrading bacteria were placed in MSM culture medium with PU plastic as the only carbon source and incubated for 60 days. The residual microplastics were collected, and physical and chemical characterization methods such as infrared spectroscopy (FTIR), thermogravimetric analysis (TGA), and gel permeation chromatography (GPC) were used to determine the structural changes of the plastics and verify the degradation ability of the bacteria on plastics. The results showed that the FTIR spectrum of residual PU in the culture medium showed the generation of new functional groups such as OH stretching vibration, CO stretching vibration and C=O stretching vibration, indicating that oxidative depolymerization of PS microplastics occurred. The thermal stability of residual PU plastics was significantly lower than that of PU microplastics at a mass loss of 5%. In addition, the molecular weight of residual PU plastics also decreased significantly. These characterization results demonstrated the structural changes of microplastics after incubation, verified the biodegradation of PU microplastics, and proved the reliability and universality of this method.

[0067] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present application. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0068] In addition, the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include at least one of the features. In the description of this application, the meaning of "plurality" is at least two, such as two, three, etc., unless otherwise clearly and specifically defined.

Claims

1. A method for screening and culturing active plastic-degrading bacteria based on Raman spectroscopy combined with heavy water labeling, characterized in that: The following steps are involved: S1. The extracted microorganisms were added to MSM liquid medium containing heavy water and PS microplastics for incubation. At the same time, intestinal microorganisms without microplastics, Escherichia coli with PS, and Staphylococcus aureus with PS were used as negative controls, and the screened PS-degrading bacteria Pseudomonas aeruginosa-IUE with PS were used as positive controls. After incubation under the same conditions, Raman detection was performed; S2. Calculate CD / (C-D+CH) of the treatment group and the control group respectively, and use three times the average value of the negative control group plus three times the standard deviation as the discrimination criterion, and set it as a threshold with statistical difference for the detection and sorting of highly active microorganisms that degrade polystyrene microplastics. A value less than or equal to 25.72 is judged as having no degradation ability for PS plastics, and a value greater than or equal to 25.72 is judged as PS highly active plastic degrading bacteria.

2. The method for screening and culturing active plastic-degrading bacteria based on Raman spectroscopy combined with heavy water labeling according to claim 1, characterized in that: The microorganisms in step S1 are extracted from the intestines of mealworm larvae fed with polystyrene foam as the only food for 42 days, and the extraction method is: S11. Pick about 10 mealworms and soak them in 75% ethanol for 5 minutes; S12, after being filled with sterile ultrapure water for 5 min, the intestine of mealworms was dissected on a sterile wax tray and placed in a 1.5 mL sterile centrifuge tube; S13, add 200 μL sterile PBS phosphate buffer and homogenize with a homogenizer; S14. The formula of PBS phosphate buffer is: Na2HPO4 1.44g, KH2PO4 0.24g, NaCl 8g, KCl 0.2g, H2O1L.

3. The method of claim 2 for rapid screening, identification and cultivation of highly active plastic-degrading bacteria in the intestine of mealworms based on Raman spectroscopy combined with heavy water labeling, characterized in that: The extracted microbial homogenate is used to collect intestinal microorganisms in the intestinal homogenate by iohexol density gradient centrifugation. The collected intestinal microbial cells are washed three times by centrifugation with sterile ultrapure water and then added into culture medium for incubation. The centrifugation conditions are: 4°C, 6500rpm.

4. The method for screening and culturing active plastic-degrading bacteria based on Raman spectroscopy combined with heavy water labeling according to claim 3, characterized in that: The steps of collecting intestinal microorganisms by iohexol density gradient centrifugation are as follows: S101, adding the intestinal homogenate into a PBS solution containing 20% ​​Tween and vortexing vigorously for 30 min on a vortex shaker to separate the cells attached to the soil particles; S102, carefully adding an equal volume of iohexol solution to the soil suspension, followed by centrifugation at 14000 g and 4°C for 90 min; S103. Carefully aspirate the liquid layer containing microorganisms in the middle, add 10 volumes of PBS and centrifuge at 6500 rpm for 10 minutes.

5. The method for screening and culturing active plastic-degrading bacteria based on Raman spectroscopy combined with heavy water labeling according to claim 1, characterized in that: In step S1, the culture medium is an MSM liquid culture medium with 50% heavy water added, and 2 mg / mL of 200 μm particle size PS plastic is added as the sole carbon source, and the incubation time is 7 days, wherein the MSM liquid culture medium formula is: NaHPO4 2.8 g; KH2PO4 1.0 g; (NH4)2SO4 0.5 g; CaCl2·2H2O 0.001 mg; H3BO3 0.03 mg; FeSO4·7H2O 0.2 mg; MnCl2·4H2O 0.003 mg; NiCl2·6H2O 0.002 mg; Na2EDTA 0.5 mg; CoCl2·6H2O 0.02 mg; ZnSo4·7H2O 0.01 mg; Na2MoO4·2H2O 0.003 mg; H2O 1L; add 1.5% agar to the above reagents to make a solid culture medium.

6. The method for screening and culturing active plastic-degrading bacteria based on Raman spectroscopy combined with heavy water labeling according to claim 1, characterized in that: In the step 1, the cells after incubation were collected and washed three times by centrifugation with sterile ultrapure water at 6500 rpm, and 2 μL of bacterial liquid was dropped on a glass slide covered with aluminum foil and air-dried before Raman testing. Single-cell Raman spectra were collected using a LabRAM Aramis (HORIBA Jobin-Yvon) confocal microscopic Raman system equipped with a 300 grooves / mm diffraction grating. A 532 nm Nd:YAG laser was used as the excitation light and a 100x air objective lens with a numerical aperture of 0.9 (Olympus, Japan) was used to observe bacteria and collect Raman signals. The acquisition time of each Raman spectrum was 25 s, and the spectrum acquisition range was 400-3200 cm -1 .

7. The method for screening and culturing active plastic-degrading bacteria based on Raman spectroscopy combined with heavy water labeling according to claim 1, characterized in that: The highly active polystyrene-degrading functional microorganisms identified by Raman were laser-ejected and sorted using a single-cell sorter. The bacteria were sorted into a receiver containing 3 μL PBS. About 20-30 single cells were collected during each sorting process. Subsequently, multiple displacement amplification was performed using the genome amplification kit REPLI-g Single Cell Kit according to the instructions, and finally the amplified products were subjected to metagenomic sequencing analysis.

8. The method for screening and culturing active plastic-degrading bacteria based on Raman spectroscopy combined with heavy water labeling according to claim 1, characterized in that: The residual microplastics were recovered after incubation in the culture medium for 30 days, ultrasonically cleaned and dried, and then the structural changes of the microplastics after incubation were determined by scanning electron microscopy, water contact angle, gel permeation chromatography, infrared spectrometer, thermogravimetric analysis, and high-performance liquid chromatography-mass spectrometry to verify the biodegradation of PS microplastics.

9. The method for screening and culturing active plastic-degrading bacteria based on Raman spectroscopy combined with heavy water labeling according to claim 6, characterized in that: The flow Raman instrument FlowRACS was used to detect, screen and sort out highly active plastic-degrading bacteria. The sorted cells were cultured on LB plates for 2 days, and then the harvested cells were placed in liquid MSM culture medium supplemented with 50 mg of PS microplastics and cultured at 30°C for 30 days. The culture was then diluted and spread on MSM agar medium with PS particles as the sole carbon source. Pure colonies were screened and the primer pair 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3') were used to amplify the nearly full-length 16S rRNA gene sequence to determine its species classification status. The obtained plastic-degrading bacteria were placed in MSM culture medium with PS plastic as the sole carbon source and incubated for 60 days. The residual microplastics were collected and the structural changes of the plastics were determined by infrared spectroscopy, thermogravimetric analysis and gel permeation chromatography, which were physical and chemical characterization methods, to verify the bacteria's ability to degrade plastics.

10. The method for screening and culturing active plastic-degrading bacteria based on Raman spectroscopy combined with heavy water labeling according to claim 9, characterized in that: The formula of LB solid culture medium is: tryptone 10g; yeast extract 5g; NaCl 10g; H2O1L; agar 15g.

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