Screening method of plastic degrading bacteria
By screening Bacillus subtilis and Bacillus cereus from the intestines of the greater wax moth and using their enzymes to degrade plastics, the problem of microplastics being difficult to degrade was solved, achieving efficient and low-cost biodegradation.
Patent Information
- Application Number
- CN202510989622.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-17
- Publication Date
- 2025-10-17
AI Technical Summary
Existing technologies are difficult to effectively degrade plastic waste, especially microplastics, which leads to serious environmental pollution. Traditional degradation methods are costly and have a great environmental impact.
Bacillus subtilis and Bacillus cereus with plastic-degrading ability were screened out from the intestinal tissue of the greater wax moth that gnawed on PE film. By co-culturing with PE film, the biodegradation of plastic was achieved using enzymes such as laccase, peroxidase, and cutinase.
The strains with high-efficiency plastic degradation ability were successfully screened out, with significant degradation effect, providing a new green and environmentally friendly microplastic degradation solution with high degradation efficiency and low cost.
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Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the field of microbial technology, in particular to a screening method of plastic-degrading bacteria. BACKGROUND
[0002] Plastic products have been widely used in modern society due to their unique advantages. However, the use of plastic products in large quantities inevitably produces plastic waste. Since plastic is a high molecular polymer, it is stable in nature and difficult to degrade, and the recycling rate is low, resulting in excessive accumulation of microplastics, causing environmental deterioration and seriously affecting people's daily life and production activities. Microplastics are divided into primary microplastics and secondary microplastics. Generally speaking, when rivers, sewage treatment plants and the like directly discharge plastic particle products, the plastic particles discharged into the water environment are called primary microplastics. When plastic waste is continuously corroded, aged, oxidized and degraded through a series of processes (such as physical, chemical and biological processes), the plastic particles formed by the splitting of plastic are called secondary microplastics. Under the action of light, friction, etc., the plastic particles formed by the fragmentation of plastic bags discarded in the water body are common secondary microplastics. Secondary microplastics are the main culprit of microplastic pollution in the water environment.
[0003] Polyethylene (PE): The structural formula of PE is —[—CH2—CH2—] n As can be seen from the structural formula, PE is a thermoplastic resin obtained by polymerization of ethylene, and people can choose different polymerization methods to obtain various functional PE. PE is one of the most widely used synthetic polymer materials in the world, with the largest production. Due to its good insulation, stability, low temperature resistance, easy molding, acid and alkali corrosion resistance and other characteristics, PE is widely used in various fields, such as building packaging, daily necessities, film and the like. PE is also the most common petroleum-based plastic. However, the service life of PE products is very short, and people use a large amount of PE products and discard them in a short period of time, resulting in a large accumulation of PE waste in the environment.
[0004] Currently, the main methods for treating plastic waste are incineration, landfill, chemical degradation and recycling. However, these non-biodegradable methods all have many limitations and are not ideal degradation methods for plastics. The research on microplastic degradation has become one of the hotspots in the field of environmental research. With the exploration of microplastic degradation methods, microplastics have received more and more attention. Biodegradation of microplastics is a new green and environmentally friendly solution, which has lower cost and causes less environmental pollution compared to other degradation methods. SUMMARY
[0005] Therefore, the application aims to provide a screening method of plastic-degrading bacteria, which can successfully screen bacteria strains with microplastic-degrading ability and provide new strains for microplastic degradation.
[0006] In order to achieve the above-mentioned purpose, the application provides the following technical solutions.
[0007] A screening method of plastic-degrading bacteria, the screening method is to screen candidate plastic-degrading bacteria with plastic-degrading ability from the intestinal tissue of Galleria mellonella which eats PE film; the candidate plastic-degrading bacteria is co-cultured with PE film, and the PE film appears perforation, and can secrete plastic-degrading related enzymes, which are plastic-degrading bacteria; the plastic-degrading related enzymes are one or several of laccase, peroxidase and cutinase;
[0008] The plastic-degrading bacteria are one or both of Bacillus subtilis and Bacillus cereus.
[0009] In the application, the following steps are specifically included:
[0010] (1) screening Galleria mellonella which eats PE film;
[0011] (2) preparing bacterial suspension: taking 15-25 Galleria mellonella screened in (1), after surface disinfection, the intestinal contents are dissolved in sterile water, constant-temperature oscillation, and dispersed into bacterial suspension;
[0012] (3) gradient dilution of bacterial suspension: using sterile water to gradient dilute the bacterial suspension to 10 -2 ~10 -6 ;
[0013] (4) inoculation and culture: using spread culture method to spread the bacterial suspension on beef extract peptone solid culture medium, and constant-temperature culture at 37 DEG C for 24-48 h;
[0014] (5) screening bacteria strains with microplastic-degrading ability:
[0015] (4) The bacteria after isolation and purification are spread on solid culture medium, and sterilized PE film is placed on the surface, and 37 DEG C enrichment culture is carried out for 15 d, and the state of PE film is observed, and the strain with perforation is the candidate plastic-degrading bacteria;
[0016] (6) drawing the growth curve of the candidate plastic-degrading bacteria, determining the optimal nitrogen source and nitrogen source; under the conditions of the optimal carbon source and nitrogen source, logarithmic phase bacterial suspension of the candidate plastic-degrading bacteria is inoculated, oscillation culture is carried out for 64 h, the activity of plastic-degrading related enzymes is detected, and the plastic-degrading bacteria are analyzed and screened;
[0017] (7) Verification: the mixed bacteria and single strain of plastic-degrading bacteria are respectively co-cultured with the PE microplastics, the weight change of the PE microplastics is measured, and the weight loss rate of the PE microplastics is calculated.
[0018] In the present application, the method of screening in (1) is preferably placing the Galleria mellonella in a PE film container only, the relative humidity is 70%-80%, the rearing temperature is 25-30℃, and the Galleria mellonella is bred for 30-35 days.
[0019] In the present application, the surface disinfection in (2) is preferably soaking the Galleria mellonella in alcohol with a volume concentration of 75% for 2-5 minutes, and oscillating at a constant temperature of 150-180 r / min for 10-20 minutes.
[0020] In the present application, the size of the PE film in (5) is preferably 2 cm x 2 cm.
[0021] In the present application, the inoculation amount in (6) is preferably 1%.
[0022] In the present application, the co-culture in (7) is preferably co-cultured in a basic inorganic salt medium with sterilized PE microplastics as the only carbon source; the co-culture is preferably at 28℃ for 64 hours; before the co-culture, the bacterial suspension is preferably centrifuged, washed, and resuspended, the centrifugation is preferably at 4500 rpm for 5 minutes, the washing is preferably with sterile water for 3 times, and the resuspension is preferably with sterile water.
[0023] In the present application, the determination of the weight change of the PE microplastics in (7) is preferably every 8 hours. Eight 250 mL conical flasks are taken, inoculated with 1% of the plastic-degrading bacteria in a basic inorganic salt medium with sterilized PE microplastics as the only carbon source, and co-cultured with the bacterial strain and the microplastics. Every 0h, 8h, 16h, 24h, 32h, 40h, 48h, and 56h, the microplastics are taken out for weight determination.
[0024] In the present application, the calculation method of the weight loss rate of the PE microplastics in (7) is preferably: the weight loss rate of the PE microplastics = (the added amount of the PE microplastics - the mass after degradation of the PE microplastics) / the added amount of the PE microplastics x 100%.
[0025] In the present application, the drawing of the growth curve of the candidate plastic-degrading bacteria in (6) preferably includes the drawing of the growth curve of the strain under different culture times, the drawing of the growth curve under different nitrogen sources, and the drawing of the growth curve under different carbon sources. The present application understands the growth rule of the strain and determines the optimal culture time and active stage by drawing the growth curve.
[0026] In the present application, the growth curve drawing of the strain under different culture times is specifically as follows: picking colonies, inoculating in beef extract protein peptone liquid medium, constant temperature shaking culture, measuring the absorbance value of the bacterial liquid at 600 nm wavelength every 1 h, and drawing the growth curve. The constant temperature shaking culture is preferably 37℃, 160r / min constant temperature shaking culture.
[0027] In the present application, the growth curve drawing under different nitrogen sources is specifically as follows: taking protein peptone, ammonium sulfate and urea as nitrogen sources respectively, weighing the required weight of beef extract (3.0g), protein peptone (5.0g) and sodium chloride (5.0g), putting the ingredients into 1000mL distilled water, stirring until completely dissolved. Using pH paper or pH meter, measuring the pH value of the medium solution. If the pH value is lower than 7.0, NaOH solution is added to adjust to pH 7.2-7.4. If the pH value is higher than 7.4, HCl solution is added to adjust to pH 7.2-7.4, to prepare beef extract protein peptone liquid medium, inoculate bacterial suspension, shake culture for 12h-14h, measure the absorbance value of the bacterial liquid at 600 nm wavelength every 4h, and draw the growth curve. The present application explores the utilization of different nitrogen sources by drawing the growth curve under different nitrogen sources, finds the most suitable nitrogen source to optimize the culture conditions, and improves the plastic degradation efficiency.
[0028] In the present application, the protein peptone and ammonium sulfate are preferably 1g-3g per 100mL beef extract protein peptone liquid medium, and the urea is preferably 1mL-3mL per 100mL beef extract protein peptone liquid medium; the urea is preferably 1% urea aqueous solution.
[0029] In the present application, the growth curve drawing under different carbon sources is preferably as follows: taking PS, PE and PET as the only carbon source of plastic degrading bacteria respectively, configuring three 100mL beef extract protein peptone liquid medium, adding 0.1%(m / v) PS, PE and PET, sterilizing in a high-pressure sterilization pot for 20min-40min, inoculating 3mL-10mL bacterial liquid, 37℃, 160r / min shaking culture, measuring the OD 600 of the bacterial suspension every 4h, and drawing the growth curve.
[0030] Beneficial technical effects: the present application provides a screening method of plastic degrading bacteria, the screening method is to screen candidate plastic degrading bacteria with plastic degrading ability from the intestinal tract tissue of the galleria mellonella which eats PE film; the candidate plastic degrading bacteria is co-cultured with PE film, the PE film appears perforation, and the plastic degrading related enzyme can be secreted, which is the plastic degrading bacteria; the plastic degrading related enzyme is one or several of laccase, peroxidase and cutinase; the plastic degrading bacteria is one or both of bacillus subtilis and bacillus cereus. The screening method of the present application can successfully screen the strain with plastic degrading ability, through the degradation characteristic research of the obtained strain, the present application can provide new strains for plastic degradation. BRIEF DESCRIPTION OF DRAWINGS
[0031] Figure 1 The galleria mellonella is fed in the feeding environment with PE film as the only food source;
[0032] Figure 2 The galleria mellonella is fed in the feeding environment with PE film as the only food source;
[0033] Figure 3 The BS2 strain solid screening culture result for 15d;
[0034] Figure 4 The BE5 strain solid screening culture result for 15d;
[0035] Figure 5 The gram staining result graph of the BS2 strain;
[0036] Figure 6 The gram staining result graph of the BE5 strain;
[0037] Figure 7 The growth curve of mixed bacteria and single strain under different culture time;
[0038] Figure 8 The growth curve of mixed bacteria under different nitrogen sources;
[0039] Figure 9 The growth curve of mixed bacteria under different carbon sources;
[0040] Figure 10 The laccase enzyme activity change of mixed bacteria and single strain;
[0041] Figure 11 The lignin peroxidase enzyme activity change of mixed bacteria and single strain;
[0042] Figure 12 The cutinase enzyme activity change of mixed bacteria and single strain;
[0043] Figure 13The weight loss rate of PE microplastics under different culture times of mixed bacteria and single strain. DETAILED DESCRIPTION
[0044] In order to better understand the present application, the content of the present application is further illustrated below in combination with examples, but the content of the present application is not limited only to the following examples. The materials, reagents and the like used in the examples and test examples of the present application can be obtained from commercial channels unless otherwise specified; the methods used in the examples and test examples of the present application are conventional methods unless otherwise specified.
[0045] Example 1
[0046] 1. Materials
[0047] 1.1 Galleria mellonella source
[0048] The Galleria mellonella used in this experiment was 2-3 instar larvae, 10-20 mm in length, purchased from Tianjin Huixuode Biological Technology Co., Ltd.
[0049] 1.2 Polyethylene plastic
[0050] (1) PE film: material is linear low density polyethylene (Linear low density polyethylene, abbreviated as LLDPE), thickness is 0.5 x 10 -3 cm, purchased from Linyi Huashuo Plastic Film Factory in China; PE microplastics were purchased from Dongguan Plastic Raw Material Co., Ltd.
[0051] 2. Methods
[0052] 2.1 Selection of Galleria mellonella feeding PE film:
[0053] Cut the PE film into 2 cm x 2 cm square pieces, sterilize with 75% ethanol and dry in a clean bench. Put 150-250 live Galleria mellonella into several insect breeding boxes, and feed them with PE film, wheat bran, and PE film + wheat bran respectively. The feeding conditions are as follows: temperature 26-28℃, relative humidity 50-70%, 12h light and 12h dark cycle, and feeding for 31d.
[0054] 2.2 Preparation of bacterial suspension: select 15-25 Galleria mellonella feeding polyethylene from the polyethylene feeding group in the breeding box, soak in alcohol for 2-5 min, dissect, and dissolve the intestinal contents of Galleria mellonella in 100 mL sterile water, put into a constant temperature oscillator, oscillate at 160 r / min for 10-20 min.
[0055] 2.3 Gradient dilution: take 0.5 mL bacterial suspension with a sterile pipette and place it in 4.5 mL sterile water, blow the pipette 3 times, mix for 5-10 min, and then it is 10-1 The mixed solution was diluted to 10 -2 ~ 10 -6 .
[0056] 2.4 Inoculation and culture: Pour beef extract peptone agar medium into a culture dish, after solidification, drop 0.1 mL ~ 0.5 mL of the diluted solution evenly, cover, and place in a constant temperature incubator for 24 h ~ 48 h, then take out, separate and purify.
[0057] 2.5 Screening of strains with plastic degradation ability
[0058] Use an inoculation loop to pick different colonies, streak on a solid medium plate, and place a 2 cm x 2 cm sterilized PE film on the surface of the solid medium, perform enrichment culture, and after streaking, place the culture dish in a constant temperature incubator at 37°C for 15 d, then take out the culture dish and observe the state of the colonies and the PE film.
[0059] 2.6 Drawing a growth curve
[0060] 2.6.1 Effect of different culture times on the strain:
[0061] Pick the colonies and dissolve in 100 mL beef extract peptone medium, place in a constant temperature shaker at 150 r / min ~ 180 r / min, shake and culture, measure the OD 600 of the bacterial solution every 1 h, and draw a growth curve.
[0062] 2.6.2 Effect of different carbon sources on the strain:
[0063] Measure the OD 600 under different carbon sources: Use PS, PE and PET as the only carbon source for plastic degrading bacteria, respectively, configure three 100 mL beef extract peptone liquid media, add 0.1% (m / v) of PS, PE and PET, sterilize in a high pressure sterilization pot for 20 min ~ 40 min, inoculate 3 mL ~ 10 mL of bacterial solution, shake and culture at 37°C and 160 r / min, measure the OD 600 of the bacterial suspension every 4 h, and draw a growth curve.
[0064] 2.6.3 Effect of different nitrogen sources on the strain:
[0065] Use peptone, ammonium sulfate and urea as the nitrogen source of the medium, respectively, configure three 100 mL beef extract peptone liquid media, add 1 g ~ 3 g of peptone and ammonium sulfate, and 1 mL ~ 3 mL of urea, sterilize in a high pressure sterilization pot for 20 min ~ 40 min, inoculate 3 mL ~ 10 mL of bacterial solution, constant temperature shake culture, measure the OD 600 of the strain under different nitrogen sources every 4 h with a spectrophotometer, and draw a growth curve.
[0066] 2.7 The characteristics and detection methods of plastic degradation related enzyme activities
[0067] Based on the growth curve in 2.6, the optimal carbon source and nitrogen source were determined; under the conditions of the optimal carbon source and nitrogen source, logarithmic phase bacterial suspension of the candidate plastic degrading bacteria was inoculated, and the activity of the plastic degradation related enzyme was detected after 64h of shaking culture, so as to analyze and screen the plastic degrading bacteria.
[0068] 2.7.1 Laccase
[0069] Detection method: ABTS method (most commonly used): reagents and instruments: ABTS (2, 2'-azino-bis (3-ethyl-benzothiazoline-6-sulfonic acid) diammonium salt), UV-2100 type ultraviolet spectrophotometer.
[0070] Reaction system: 1.8mL acetic acid-sodium acetate buffer (50mmol / L, pH 4.0) + 0.8mL ABTS (1mmol / L) + 0.4mL enzyme solution, 30℃ reaction for 3min.
[0071] Calculation formula: enzyme activity (U / L) = A x n x 13.89 (A is absorbance, n is dilution multiple, and 13.89 is the coefficient).
[0072] 2.7.2 Lignin peroxidase
[0073] Detection method: veratryl alcohol oxidation method: the enzyme activity is calculated by measuring the change in absorbance of veratraldehyde at 310nm. The reaction system includes veratryl alcohol, tartaric acid buffer (pH 3.0) and H2O2.
[0074] 2.7.3 Cutinase
[0075] Detection method: colorimetric method: using substrates such as ester compounds, cutinase catalyzing hydrolysis to generate soluble products, and measuring the absorbance to calculate the enzyme activity.
[0076] 2.8 Changes in weight loss rate of PE microplastics at different times
[0077] BS2 and BE 5 separated and purified were respectively cultured to the logarithmic growth phase, 20mL bacterial suspension was centrifuged at 4500r / min for 5min, washed with sterile water for 3 times, then 6mL sterile water was added for resuspension, and the resuspended bacterial solution was added to the basic inorganic salt medium (100mL) with 0.2g sterilized PE plastic as the only carbon source, and cultured at 150r / min and 28℃ for a total of 64h. The PE microplastics were recovered, and the mass change before and after degradation in single strain and mixed bacteria was measured every 8h to evaluate the microplastic degradation ability of microorganisms.
[0078] The weight loss rate of the PE film = (the added amount of PE microplastics - the mass of PE microplastics after degradation) / the added amount of PE microplastics * 100%.
[0079] 3. Results and analysis
[0080] 3.1 Screening results of Galleria mellonella insects that eat PE film
[0081] The survival rates of Galleria mellonella under different feeding modes are shown in Table 1.
[0082] The PE film sample is in the form of a film, has a smooth surface, and is not easy to eat. After the feeding ended, a number of plastic samples were picked out for comparison before and after, and it was found that many holes appeared on the surface of the plastic, and the feeding traces of Galleria mellonella were obvious, as shown in Figures 1-2 .
[0083] Table 1 Feeding mode and survival rate of Galleria mellonella
[0084]
[0085] 3.2 Screening results of strains with microplastic degradation ability
[0086] Through screening, two different morphological colonies were found, which caused the surface of the plastic film to appear perforated, as shown in Figures 3-4 . The intestinal microbial colonies of Galleria mellonella that eat polyethylene plastic were named "BS2" and "BE5", respectively. The two strains were subjected to gram staining, as shown in Figure 5 and Figure 6 . Under an oil lens, they were all purple, which were gram-positive bacteria, and were bacilli. Through physiological and biochemical identification, strains "BS2" and "BE5" were Bacillus subtilis and Bacillus cereus, respectively. As shown in Table 1.
[0087] Table 1 Screening results of colonies
[0088]
[0089] 3.3 Growth curve drawing results
[0090] The change trend of OD 600 of the bacterial suspension under different carbon sources, the OD 600 of the plastic-degrading bacteria under different carbon sources all reached the maximum at the 48th hour, and when the two were mixed, the growth curve was the best. As shown in Figure 7 .
[0091] As shown in Figure 8 , the change trend of OD 600 of the mixed bacterial suspension under different nitrogen sources, the OD 600The time to reach the maximum value was 40h, 48h and 44h. The plastic-degrading bacteria using ammonium sulfate as nitrogen source grew fastest, which indicated that the strain utilized ammonium sulfate better than peptone and urea.
[0092] Figure 9 OD of the bacterial suspension of plastic-degrading bacteria under different carbon sources 600 The changing trend of OD of plastic-degrading bacteria under different carbon sources 600 All reached their maximum at 48 hours. Among them, PE grew the fastest, had the longest growth cycle, and was the easiest to degrade, followed by PS. PET was relatively difficult to degrade. This may be related to the molecular structures of the three plastics. PE is obtained by polymerization of ethylene and has a simple structure and is easily degraded. PS is a polymer synthesized with styrene as the main raw material. PET is produced by transesterification of dimethyl terephthalate with ethylene glycol or by esterification of terephthalic acid with ethylene glycol to form dihydroxyethyl terephthalate, followed by polycondensation. It has the highest hardness and is the most difficult to degrade.
[0093] 3.4 Changes in enzyme activity at different times
[0094] Under optimal carbon and nitrogen source conditions, enzyme production by mixed and individual strains was tested. Laccase, lignin peroxidase, and cutinase, which are involved in plastic degradation, were measured for both mixed and individual strains over time. Microorganisms can also secrete a variety of enzymes during their life processes that contribute to the biodegradation of microplastics.
[0095] Enzymatic biodegradation of plastics typically involves two steps: first, the enzyme binds to the plastic through its active site; then, the enzyme degrades the plastic under the catalytic action of the active site. Cracking and depolymerization are key steps in microplastic biodegradation. The enzymes involved in microplastic degradation primarily include hydrolases, amidases, monooxygenases, peroxidases (manganese peroxidase, soybean peroxidase, glutathione peroxidase), laccases, and cutinases. Currently, the enzymes that can degrade plastics are primarily hydrolases and peroxidases. Among these, extracellular hydrolases secreted by microorganisms are primarily involved in the degradation of microplastic polymers and the cleavage of bonds (such as ─COOR and ─CONH). Currently, the enzymes discovered to degrade polyethylene (PE) are primarily laccases and alkane hydroxylases. Laccases and manganese peroxidases can undergo terminal oxidation, while the AlkB (AlkB enzyme: alkane monooxygenase) family can degrade n-alkanes, the main component of PE, through terminal or subterminal hydroxylation reactions.
[0096] Laccase activity at different times Figure 10 As shown, the laccase activity of BE 5 reached its maximum at 24 h, which was 37.95 U / L; the laccase activity of BS2 reached its maximum at 32 h, which was 35.60 U / L; and the laccase activity of the mixed bacteria reached its best at 40 h, which was 49.65 U / L.
[0097] Peroxidase enzyme activity at different times Figure 11 As shown in the table, the peroxidase enzyme activity of BE 5 and BS2 reached the maximum at 24h, which were 3.50U / L and 4.20U / L respectively; the peroxidase enzyme activity of mixed bacteria reached the optimum at 16h, which was 5.80U / L. Figure 12 As shown in the table, the cutinase enzyme activity of BE 5 reached the maximum at 40h, which was 1.60U / L; the cutinase enzyme activity of BS2 reached the maximum at 32h, which was 1.50U / L; the cutinase enzyme activity of mixed bacteria reached the optimum at 40h, which was 1.90U / L.
[0098] 3.5 Weight loss rate change of PE microplastics at different times
[0099] The PE microplastics were co-cultured with mixed bacteria and single strains respectively, and the weight change of PE microplastics was measured at different times to calculate the weight loss rate of PE microplastics. Specifically, 8 250mL conical flasks were taken, and 1% of plastic degrading bacteria was inoculated in the basic inorganic salt medium with sterilized PE microplastics as the only carbon source to carry out strain microplastic co-culture, and every 0h, 8h, 16h, 24h, 32h, 40h, 48h, 56h was taken out for microplastic weight determination.
[0100] The weight loss rate change of PE microplastics at different times is shown in the table Figure 13 As shown in the table, the weight loss rate of PE film of mixed bacteria reached 51.69% after 56h of culture, which was higher than that of BE5 (35.77%) and BS2 (39.89%), and showed good plastic degradation potential, which was a potential functional composite bacteria for PE plastic degradation.
[0101] The above only describes the preferred embodiments of the present application, and it should be noted that for those skilled in the art, without departing from the principles of the present application, a number of improvements and refinements can be made, and these improvements and refinements should also be considered as the protection scope of the present application.
Claims
1. A method for screening plastic-degrading bacteria, characterized in that: The screening method comprises isolating and screening candidate plastic-degrading bacteria with plastic-degrading ability from the intestinal tissue of a greater wax moth that gnaws a PE film; co-culturing the candidate plastic-degrading bacteria with the PE film; and if the PE film is perforated and can secrete enzymes related to plastic degradation, the candidate plastic-degrading bacteria are identified; the enzymes related to plastic degradation are one or more of laccase, peroxidase, and cutinase; The plastic degrading bacteria are one or both of Bacillus subtilis and Bacillus cereus.
2. The screening method according to claim 1, wherein The specific steps include: (1) Screening for wax moths that feed on PE films; (2) Preparation of bacterial suspension: 15 to 25 wax moths screened in (1) were taken, their surfaces disinfected, and their intestinal contents dissolved in sterile water, shaken at a constant temperature, and dispersed into a bacterial suspension; (3) Gradient dilution of bacterial suspension: Use sterile water to gradient dilute the bacterial suspension to 10 -2 ~10 -6 ; (4) Inoculation and culture: The bacterial suspension was spread on beef extract peptone solid medium using the spread culture method and cultured at 37°C for 24–48 h. (5) Screening of strains with plastic-degrading ability: The bacteria isolated and purified in (4) were spread on a solid culture medium, and sterilized PE films were placed on the surface of the culture medium. The culture was enriched at 37°C, and the state of the PE films was observed. The strains with perforations were selected as candidate plastic-degrading bacteria. (6) Further screening: draw the growth curve of candidate plastic-degrading bacteria and determine the optimal carbon source and nitrogen source; under the conditions of the optimal carbon source and nitrogen source, inoculate the logarithmic phase bacterial suspension of candidate plastic-degrading bacteria, shake culture for 64 hours, detect the activity of plastic degradation-related enzymes, and analyze and screen to obtain plastic-degrading bacteria; (7) Verification: PE microplastics were co-cultured with mixed bacteria and single strains of plastic-degrading bacteria, the weight change of PE microplastics was measured, and the weight loss rate of PE microplastics was calculated.
3. The screening method according to claim 2, characterized in that The screening method in (1) is to place the greater wax moth in a container with only PE film for breeding, with a relative humidity of 70%-80% and a breeding temperature of 25°C-30°C for 30 days-35 days.
4. The screening method according to claim 2, wherein The surface disinfection in (2) is to soak the wax moth in 75% alcohol by volume for 2 to 5 minutes, and then shake it at a constant temperature of 150 to 180 r / min for 10 to 20 minutes.
5. The screening method according to claim 2, characterized in that The size of the PE film in (5) is 2 cm×2 cm.
6. The screening method according to claim 2, wherein The inoculation amount in (6) is 1%.
7. The screening method according to claim 2, wherein The co-cultivation in (7) is co-cultivation in a basic inorganic salt culture medium with sterilized PE microplastics as the only carbon source.
8. The screening method according to claim 2, wherein The weight change of PE microplastics in (7) is measured every 8 hours.
9. The screening method according to claim 2, characterized in that The calculation method of the weight loss rate of PE microplastics in (7) is: weight loss rate of PE microplastics = (amount of PE microplastics added - mass of PE microplastics after degradation) / amount of PE microplastics added × 100%.