Polyethylene plastic mulch degrading bacteria and application thereof

Abstract

The application discloses a polyethylene plastic mulch degrading bacterium and application thereof, and belongs to the technical field of microorganisms.The polyethylene plastic mulch degrading bacterium is Pantoea conspicua CYD50-1, which is preserved in the China Center for Type Culture Collection and has a preservation number CCTCC NO: M 20231747.The polyethylene plastic mulch degrading bacterium Pantoea conspicua CYD50-1 provided by the application has a good effect on degrading polyethylene plastic mulch, after 15 days of biodegradation treatment, the surface of the polyethylene plastic mulch is rough, obvious cracks, pits and gullies are generated on the surface, and the weight loss rate of the polyethylene plastic mulch reaches 6%, thus the application provides a new strain resource for biodegradation of polyethylene plastic mulch and has a wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, and in particular relates to a strain of polyethylene plastic film degrading bacteria and its application. Background Technology

[0002] Plastics are widely used in human production and daily life, and their production is increasing daily. However, only 18% of waste plastics are recycled or reused, 24% are incinerated, and the remaining 58% end up in landfills or the natural environment. Due to a lack of proper management, more and more plastic residues remain in the natural environment. Traditional plastics are difficult to degrade in the natural environment, persist for a long time, and may release harmful substances, causing pollution and damage to the environment, seriously endangering ecosystems and animal health, including entanglement of animals, transmission of pathogens, and disruption of the food chain. The accumulation of plastic waste and related pollution has become a serious environmental and socio-economic problem.

[0003] Most research on environmental plastic pollution focuses on aquatic environments, but plastic pollution in soil ecosystems, especially agricultural soils, also deserves sufficient attention. Plastic mulching plays a vital role in agricultural activities due to its function of improving crop yield and quality. In my country's agricultural production, plastic mulch film, with polyethylene (PE) as its main component, has been widely used for over forty years. my country ranks first in the world in both the production and consumption of agricultural plastic mulch film. Large quantities of agricultural polyethylene plastic mulch film are generally piled up and scattered in fields after use. Due to the incomplete decomposition of the plastic mulch film in the fields, large amounts of plastic fragments and microparticles accumulate in the soil for decades. These accumulated film fragments not only affect the soil environment but may also enter the food chain, thus posing a potential threat to human health and the ecological environment.

[0004] Currently, there are few methods for degrading plastic mulch film, and the results are mostly unsatisfactory. Traditional methods for disposing of waste plastics include landfill, incineration, and secondary processing, but these methods cause significant environmental pollution and are costly. In contrast, microorganisms can degrade plastic mulch film without harming the environment, and are considered a safe, clean, and efficient method. Utilizing the wide distribution of microorganisms in the environment, they can effectively degrade large areas of plastic mulch film, thus microbial degradation technology for plastic mulch film is receiving increasing attention.

[0005] The process of degrading plastics using microbial strains is environmentally friendly due to its mild degradation conditions and pollution-free degradation products, making it particularly suitable for degrading plastic mulch films in farmland. Studies have found that the microorganisms that degrade polyethylene mulch films are mainly fungi and bacteria. Fungi include *Penicillium simplicissimum* YK, *Aspergillus niger*, and *Fusarium solani*. Bacteria include *Enterobacter asburiae*, *Paenibacillus*, *Acinetobacter baumannii*, and *Pseudomonas spp.* However, the effectiveness of these strains in degrading polyethylene plastic mulch films is not entirely satisfactory, and the resources of microbial strains for degrading plastic mulch films are currently limited, restricting the widespread application of microbial degradation of plastic mulch films. Furthermore, the sources of degrading bacteria are mostly seawater, soil, plastic waste, and landfills, with few reports of bacteria from kitchen waste compost. In summary, PE biodegradable resources still need to be replenished. Strains can be isolated from the actual environment to provide excellent resources for the promotion and application of biodegradable plastic mulch films. Summary of the Invention

[0006] In view of the above, the purpose of this invention is to provide a polyethylene plastic film degrading bacterium and its application, which can effectively degrade polyethylene plastic film. The strain CYD50-1 of this application was isolated from kitchen waste compost at 50℃ on the East Lake Campus of Zhejiang Agricultural and Forestry University in Hangzhou, Zhejiang Province. Sequence comparison determined its species to be *Pantoea*. The CYD50-1 strain is most closely related to the sequence of *Pantoea conspicua*, a result consistent with morphological identification, indicating that the isolated CYD50-1 strain is *Pantoea conspicua*.

[0007] To achieve the above-mentioned objectives, the technical solution adopted by the present invention is as follows:

[0008] This invention provides a polyethylene plastic film degrading bacterium, Pantoea conspicua CYD50-1, which is deposited at the China Center for Type Culture Collection, accession number CCTCC NO: M20231747.

[0009] The present invention also provides the application of polyethylene plastic film degrading bacteria in the degradation of polyethylene plastic film.

[0010] The present invention also provides the application of polyethylene plastic film degrading bacteria in the preparation of polyethylene plastic film degrading agents.

[0011] The present invention also provides a polyethylene plastic mulch film degrading agent, wherein the active ingredient comprises the above-mentioned polyethylene plastic mulch film degrading bacteria.

[0012] The present invention also provides a method for preparing a polyethylene plastic mulch film degrading agent, wherein the polyethylene plastic mulch film degrading bacteria are inoculated into a culture medium, and the bacterial solution prepared after cultivation is the polyethylene plastic mulch film degrading agent.

[0013] The present invention also provides a method for degrading polyethylene plastic mulch film, wherein the polyethylene plastic mulch film is degraded using the aforementioned polyethylene plastic mulch film degrading bacteria or the aforementioned polyethylene plastic mulch film degrading agent.

[0014] Preferably, the specific steps for using the aforementioned polyethylene plastic film degrading bacteria to degrade the polyethylene plastic film are as follows:

[0015] (1) Cut the polyethylene plastic film into small pieces;

[0016] (2) The small membrane was first soaked in sodium dodecyl sulfate solution, then ultrasonically cleaned in ethanol of the first concentration, then soaked in ethanol of the second concentration, then rinsed several times with anhydrous ethanol, and finally the small membrane was dried in an oven to obtain sterilized small membrane, with the first concentration being lower than the second concentration.

[0017] (3) Add the sterilized small membrane to the inorganic salt culture medium, prepare the polyethylene plastic film degrading bacteria strain into a bacterial solution, inoculate it into the inorganic salt culture medium with polyethylene plastic film as the only carbon source, and shake the flask for culture.

[0018] Preferably, a bacterial solution is prepared by inoculating the polyethylene plastic film degrading bacteria strain at a volume ratio of 10%.

[0019] Preferably, the shaking culture conditions are 28°C and 180 rpm.

[0020] Compared with the prior art, the beneficial effects of the present invention are:

[0021] The polyethylene plastic film degrading bacterium Pantoea conspicua CYD50-1 provided by this invention has a good effect on degrading polyethylene plastic film. After 15 days of biodegradation treatment, the surface of the polyethylene plastic film becomes rough, with obvious cracks, pits and grooves. The weight loss rate of the polyethylene plastic film reaches 6%. This provides a new strain resource for the biodegradation of polyethylene plastic film and has broad application prospects. Attached Figure Description

[0022] Figure 1 The colony morphology of the degrading bacterium CYD50-1 provided by this invention is shown in the figure.

[0023] Figure 2 Scanning electron microscope image of the degrading bacterium CYD50-1 provided by the present invention;

[0024] Figure 3 A schematic diagram of the growth curve of the degrading bacterium CYD50-1 provided by the present invention;

[0025] Figure 4 Scanning electron microscope image of PE film after 15 days of degradation by the inoculated degrading bacteria CYD50-1 provided by the present invention;

[0026] Figure 5 The image shows the scanning electron microscope features of the PE film after 15 days of degradation in the blank control group (without inoculation with the degrading bacterium CYD50-1) provided by this invention. Detailed Implementation

[0027] Example 1: Isolation and Screening of Strains

[0028] (1) Sample collection

[0029] The 50℃ kitchen waste compost was collected from the East Lake Campus of Zhejiang Agriculture and Forestry University in Hangzhou, Zhejiang Province as a sample.

[0030] (2) Preparation of microbial inoculum

[0031] Weigh 20.0g of sample and transfer it to a 500mL Erlenmeyer flask containing 180mL of sterile physiological saline. Shake at 180rpm for 30min. After standing, take the supernatant as the microbial inoculum.

[0032] (3) Material pretreatment

[0033] Polyethylene (PE) film was uniformly cut into small film pieces with a size of 4.0×4.0cm. The film pieces were soaked in 0.5% KCl solution for 1 hour, then washed 3-4 times with 100% anhydrous ethanol, and finally rinsed with sterile water. After drying in a dry, sterile petri dish, the film pieces were sterilized by irradiation under ultraviolet light for 4 hours and then dried in a drying oven at 50±1℃ to obtain sterile film pieces.

[0034] (4) Assembly of artificial micro-ecosystems

[0035] First, inorganic salt culture medium was prepared. The culture medium for isolation and screening was inorganic salt culture medium (g / L) with PE film as the sole carbon source. 180mL of inorganic salt liquid culture medium was prepared for every 500mL Erlenmeyer flask. The pH was adjusted to 7 with 1mol / L HCl and sterilized by high temperature steam at 121℃ for 30min.

[0036] Table 1. Reagents and dosage (1L) for inorganic salt culture medium

[0037]

[0038] Subsequently, a pretreated sterile membrane was inoculated into an inorganic salt culture medium to prepare a sterile artificial micro-ecosystem with PE film as the sole carbon source.

[0039] (5) Domestication of plastic-degrading bacteria

[0040] 10 mL of microbial inoculum was added to the sterile artificial micro-ecosystem described above, which used PE film as the sole carbon source. The mixture was then incubated in a shaker at 35°C and 180 rpm as the experimental group. Simultaneously, two 200 mL inorganic salt culture media were each inoculated with only one sterile film patch as a blank control group. Both groups were incubated under the same conditions, and observations were conducted daily. Inoculation was performed weekly at a rate of 10% in fresh inorganic salt liquid culture medium, repeated for several cycles. Compared to the blank control group, the experimental group's culture medium was significantly turbid. This was confirmed by measuring OD... 600 This also indicates that a bacterial strain is growing in the culture medium.

[0041] (6) Screening of plastic-degrading bacteria

[0042] Take 1 mL of the suspension from the enriched culture medium and dilute it with sterile distilled water to prepare 10 mL of solution. -1 ~10 -7 Serial dilution of bacterial culture, 10 -4 10 -6 The diluted bacterial solution was spread onto an inorganic salt solid culture medium with PE film as the sole carbon source. Each concentration was repeated three times. The culture was placed in an incubator at 30°C and incubated for 48 hours. The growth of the strain was then observed.

[0043] (7) Enrichment and purification of plastic-degrading bacteria

[0044] After distinct colonies grew on the plates, their size, color, and morphology were observed. Different strains that grew well using PE film as the sole carbon source were selected, and strains with different morphologies were marked. These strains were then streaked again onto different inorganic salt solid media and incubated at 30°C for 48 hours. The selected strains were then streaked repeatedly on the plates at least three times to purify them, obtaining single colonies. These single colonies were then streaked onto LB solid media and stored at 4°C, numbered, and stored.

[0045] Table 2. Reagents and dosages for LB culture medium (1L)

[0046]

[0047] LB solid medium is prepared by adding 18g of agar powder per liter and placing it in an incubator for 24 hours. If no abnormalities are observed, it is ready for use.

[0048] Enrichment culture using inorganic salt medium with PE film as the sole carbon source revealed that a large number of potential degrading bacteria capable of using PE film as a carbon source accumulated on the PE film. Colonies grew along the edge of the PE film, with fewer colonies growing further away from the PE film. Through enrichment culture of the colonies, a potentially highly efficient polyethylene plastic degrading bacteria strain with PE film as the sole carbon source was isolated from kitchen waste compost and named CYD50-1.

[0049] Example 2: Identification of the strain

[0050] Morphological identification:

[0051] like Figure 1 As shown, strain CYD50-1 is a golden-yellow strain with irregular colonies, a moist and smooth surface, easy to pick up, and semi-transparent. Strain CYD50-1 exhibits distinct dissolution zones on inorganic and organic phosphorus plates, and appears as spherical protrusions on nitrogen-fixing plates. Figure 2 The image shown is from a scanning electron microscope. The cells are rod-shaped, (0.5–1.0) μm × (1–2) μm, and move via peritrichous flagella. Most strains produce a yellow pigment. They are facultative anaerobic, exhibiting both metabolic and fermentative chemoheterotrophic characteristics. Based on colony morphology and molecular biological identification results, strain CYD50-1 was identified as *Pantoea conspicua*.

[0052] Molecular identification:

[0053] (1) DNA extraction

[0054] The strains grown on inorganic salt culture medium with PE film as the sole carbon source were reactivated and then molecularly identified. The 16S rDNA gene sequence of the obtained strains was amplified and sequenced, and the DNA of the isolated single strains was extracted using a kit.

[0055] (2) PCR amplification of bacterial ribosomal 16S rDNA gene

[0056] Universal primers for bacteria: PCR amplification was performed using 27F and 1492R primers. 16S rDNA sequence primers: 27F: 5'-AGAGTTTGATCCTGGCTCAG-3', 1492R: 5'-GGTTACCTTGTTACGACTT-3'.

[0057] PCR amplification reaction system: PCR amplification was carried out in a 50 μL reaction system, with 2 μL template DNA, 25 μL LMix (2×), 2 μL primer 27F, 2 μL primer 1492R, and 19 μL ddH2O.

[0058] PCR amplification conditions: The amplification program was as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 1 min, 58℃ annealing for 1 min, 72℃ extension for 2 min, for 30 cycles; and finally 72℃ extension for 10 min, and storage at 4℃.

[0059] (3) Gene sequencing and sequence analysis

[0060] The amplified products were analyzed by electrophoresis, and the strain was sent to Shanghai Sangon Biotech Co., Ltd. for sequencing. The obtained sequences were compared with existing sequences in the NCBI database using BLAST analysis, and strains with similar homology were selected. Sequence analysis confirmed that the strain belonged to the genus *Pantoea*, and its sequence was most closely related to *Pantoea conspicua*. This result was consistent with the morphological identification results, indicating that the isolated strain CYD50-1 was *Pantoea conspicua*. This newly screened strain was named *Pantoea conspicua*, strain number CYD50-1, and was deposited on September 20, 2023, at the China Center for Type Culture Collection (CCTCC), Wuhan University, China, with accession number CCTCC NO: M20231747.

[0061] Example 3: Growth curve determination of CYD50-1 strain

[0062] The experimental material used was PE film, and the plastic-degrading bacteria was strain CYD50-1. The growth curve of strain CYD50-1 was measured after 24 hours using a liquid shake-flask culture method.

[0063] Specific procedures: First, label the corresponding bacterial strain in an Erlenmeyer flask containing 200 mL of sterile LB medium. Scrape pure bacterial cultures from the LB plates and inoculate them into the sterile LB medium for 24 hours. Place the inoculated Erlenmeyer flasks in a shaker at 37°C and 170 rpm. Every 2 hours, take 4 mL of bacterial culture from the Erlenmeyer flasks, centrifuge, and add sterile physiological saline. Use uninoculated sterile physiological saline as a blank control group. Perform photoelectric turbidimetry at a wavelength of 600 nm. Finally, plot the growth curves of the corresponding bacterial strains.

[0064] like Figure 3 As shown, the population density of strain CYD50-1 increased from 0 to 1.180 from 0 to 4 hours, and then rapidly and continuously increased from 4 hours to 16 hours, growing from 1.180 to 2.159, indicating that strain CYD50-1 entered the logarithmic growth phase during this period. The growth curve began to stabilize after 16 hours, indicating that the bacterial population gradually approached the stationary phase after 16 hours of incubation.

[0065] Example 4: Determination of weight loss rate of PE film degradation by CYD50-1 strain

[0066] Aseptic treatment: The PE film is first soaked in a 2% sodium dodecyl sulfate (SDS) solution for 4 hours, then placed in 50% ethanol and ultrasonically cleaned for 30 minutes, twice. Next, it is soaked in 70% ethanol overnight, and finally rinsed multiple times with anhydrous ethanol to ensure that biofilm and impurities are completely removed. Finally, the PE film is placed in an oven and dried at 40℃.

[0067] Culture Experiment: 4.0 × 4.0 cm PE film fragments were numbered and accurately weighed initially. After sterilization, they were added to a basic inorganic salt liquid culture medium, with a volume of 100 mL / 250 mL Erlenmeyer flask. CYD50-1 strain was inoculated at a 10% (v / v) ratio with the pre-prepared bacterial suspension, then inoculated into 100 mL of liquid inorganic salt culture medium with the corresponding PE film as the sole carbon source. Each strain was prepared in triplicate as an experimental group. The culture was carried out at 28°C and 180 rpm with shaking for 15 days. A control group (PE film-liquid inorganic salt culture medium without bacterial suspension) was cultured under the same conditions.

[0068] Calculation of PE film weight loss rate: The weight loss rate of different PE films after degradation by different test strains was calculated by weighing method. After 15 days of cultivation, small PE film pieces were taken out and sterilized, and weighed using a balance with a strength of 0.0001%. Degrading strains were screened out by calculating the degradation rate of PE film. The percentage loss before and after plastic degradation was used to characterize the degradation effect of different plastics. PE film weight loss rate = (initial PE film weight - PE film weight after degradation) / initial PE film weight.

[0069] Experimental results: After 15 days of cultivation, the weight loss rate of PE film in the experimental group and the blank control group is shown in Table 3. The average weight loss rate of the three experimental groups was 6.37%, which shows that the weight loss rate of polyethylene plastic film degraded by CYD50-1 strain can reach 6%, which has a good degradation effect.

[0070] Table 3 Weight loss rate of PE film after 15 days of cultivation.

[0071]

[0072] Example 5: Scanning electron microscopy observation of PE film degradation by strain CYD50-1

[0073] Aseptic treatment: The PE film is first soaked in a 2% sodium dodecyl sulfate (SDS) solution for 4 hours, then placed in 50% ethanol and ultrasonically cleaned for 30 minutes, twice. Next, it is soaked in 70% ethanol overnight, and finally rinsed multiple times with anhydrous ethanol to ensure that biofilm and impurities are completely removed. Finally, the PE film is placed in an oven and dried at 40℃.

[0074] Culture Experiment: 4.0 × 4.0 cm PE film fragments were numbered and accurately weighed initially. After sterilization, they were added to a basic inorganic salt liquid culture medium, with a volume of 100 mL / 250 mL Erlenmeyer flask. CYD50-1 strain was inoculated at a 10% (v / v) ratio with the pre-prepared bacterial suspension, then inoculated into 100 mL of liquid inorganic salt culture medium with the corresponding PE film as the sole carbon source. Each strain was prepared in triplicate as an experimental group. The culture was carried out at 28°C and 180 rpm with shaking for 15 days. A control group (PE film-liquid inorganic salt culture medium without bacterial suspension) was cultured under the same conditions.

[0075] Scanning electron microscope (SEM) observation: A small PE film sheet cultured for 15 days was removed. After removing impurities from the PE film surface, a small piece of film was carefully cut with scissors and attached to the sample stage using conductive adhesive. After fixing and sputtering with gold for 120 seconds, it was removed and placed into the sample chamber. The vacuum switch was turned on, and the vacuum pump started working, evacuating the air from the electron chamber and sample chamber. When a complete vacuum was achieved, the light outside the sample chamber would turn blue. The SEM software on the computer was then turned on. Based on the conductivity of the sample and the height of the sample stage, the working voltage and working distance were determined before observation began. First, the sample stage was located and centered. The magnification was gradually increased from 500x, while simultaneously adjusting the image clarity. Wrinkles and cracks in the PE film were located, and the images were saved.

[0076] Experimental results: After 15 days of degradation, the PE film in the experimental group inoculated with the degrading bacteria CYD50-1 showed the following characteristics: Figure 4 As shown, its surface is rough, with obvious cracks and pits. The blank control group, which was not inoculated with the degrading bacteria CYD50-1, degraded for 15 days, and the PE film showed... Figure 5 As shown, its surface is smooth and flat. Experimental results indicate that the PE film underwent biodegradation in the experimental group inoculated with the degrading bacteria CYD50-1.

Claims

1. A polyethylene plastic mulch degrading bacteria, characterized in that, The polyethylene plastic film degrading bacteria is Pantoeaconspicua CYD50-1, which is deposited at the China Center for Type Culture Collection, accession number CCTCC NO: M 20231747.

2. The application of the polyethylene plastic film degrading bacteria as described in claim 1 in the degradation of polyethylene plastic film.

3. The application of the polyethylene plastic film degrading bacteria as described in claim 1 in the preparation of a polyethylene plastic film degrading agent.

4. A polyethylene plastic mulch degrading agent characterized by, The active ingredient comprises the polyethylene plastic film degrading bacteria as described in claim 1.

5. A method for preparing the polyethylene plastic film degrading agent as described in claim 4, characterized in that, The polyethylene plastic film degrading bacteria described in claim 1 are inoculated into a culture medium, and the resulting bacterial solution is prepared as a polyethylene plastic film degrading agent.

6. A method for degrading polyethylene plastic mulch film, characterized in that, The polyethylene plastic mulch film is degraded using the polyethylene plastic mulch film degrading bacteria of claim 1 or the polyethylene plastic mulch film degrading agent of claim 4.

7. The method for degrading polyethylene plastic mulch film according to claim 6, characterized in that, The specific steps for using the polyethylene plastic film degrading bacteria described in claim 1 to degrade polyethylene plastic film are as follows: (1) Cut the polyethylene plastic film into small pieces; (2) The small membrane was first soaked in sodium dodecyl sulfate solution, then ultrasonically cleaned in ethanol of the first concentration, then soaked in ethanol of the second concentration, then rinsed several times with anhydrous ethanol, and finally the small membrane was dried in an oven to obtain sterilized small membrane, with the first concentration being lower than the second concentration. (3) Add the sterilized small membrane to the inorganic salt culture medium, prepare the polyethylene plastic film degrading bacteria strain into a bacterial solution, inoculate it into the inorganic salt culture medium with polyethylene plastic film as the only carbon source, and shake the flask for culture.

8. The method for degrading polyethylene plastic mulch film according to claim 7, characterized in that, A bacterial solution was prepared by inoculating a strain of bacteria that degrades polyethylene plastic film at a volume ratio of 10%.

9. The method for degrading polyethylene plastic mulch film according to claim 7, characterized in that, The shaking culture conditions were 28℃ and 180 rpm.

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