Geotrichum candidum and application of geotrichum candidum in degradation of polyethylene plastic mulching film

Abstract

The invention discloses geotrichum candidum and application thereof in degradation of a polyethylene plastic mulching film, and relates to the technical field of microorganisms and application thereof. The strain number of the Geotrichum candidum is MY-0603, the Geotrichum candidum is preserved in the China Center for Type Culture Collection, and the preservation number of the Geotrichum candidum is CCTCC (China Center for Type Culture Collection) NO: M2025455. Experimental results show that the screened strain shows excellent performance in the aspect of biodegradation of polyethylene plastic, and after 21 days of biodegradation treatment, the weight loss rate of the polyethylene plastic mulching film reaches 8%. The method has the advantages of being environmentally friendly, low in cost, easy and convenient to operate and the like, and the obtained strain has the efficient degradation characteristic. Therefore, the invention provides a new strain resource and thought for bioremediation of polyethylene waste in the environment, and has a wide application prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms and their applications. More specifically, the present invention relates to a Geotrichum candidum and its application in degrading polyethylene plastic mulch films. Background Art

[0002] In the ocean, plastic waste accounts for 85% of the total weight of marine waste, and microplastics are spread to global sea areas through ocean currents and even appear near the poles. In addition, microplastics also pose a potential threat to marine organisms and human health through the food chain.

[0003] Polyethylene (PE) plastics are widely used in various fields due to their excellent physical and chemical properties, but their difficult-to-degrade characteristics have led to environmental pollution problems. Traditional plastic treatment methods such as incineration and landfilling not only occupy a large amount of land resources but also release harmful gases, exacerbating environmental pollution. Therefore, developing an efficient and environmentally friendly polyethylene degradation technology has important practical significance.

[0004] In recent years, microbial degradation has received extensive attention as a green and sustainable solution. Research shows that a variety of bacteria and fungi can degrade polyethylene plastics, among which Pseudomonas (such as the literature: Wang Xixi, Qu Changfeng, Wang Wenyu, etc. Research status and prospects of microplastic pollution in the Chinese ocean [J]. Marine Sciences, 2018, 42(3): 11.), Bacillus (such as the literature: Liu Xianrui, Chemical Engineering and Technology. Study on the degradation effect and degradation genes of Bacillus velezensis on polyethylene [D].[2025-03-29]), Rhodococcus and other microorganisms show good degradation ability. These microorganisms secrete extracellular enzymes such as laccase and peroxidase to break the chemical bonds of polyethylene, reducing its molecular weight and thus achieving degradation. However, most of the currently screened degradation strains have problems such as low degradation efficiency and harsh degradation conditions, making it difficult to meet the actual application requirements. Summary of the Invention

[0005] The purpose of the present invention is to provide a highly efficient polyethylene plastic-degrading bacterium and its application in polyethylene plastic biodegradation. The strain of the present invention was isolated from the surface of plastic samples in the sewage of the Donghu Campus of Zhejiang A&F University in Hangzhou, Zhejiang Province. Through sequence comparison, its genus was determined to be Geotrichum, and the strain MY-0603 was found to be most closely related to Geotrichum candidum in terms of sequence. This result is the same as the morphological identification result, indicating that the isolated strain MY-0603 is Geotrichum candidum.

[0006] To achieve the above purpose, the specific technical solutions of the present invention are as follows:

[0007] The present invention provides a Geotrichum candidum, named Geotrichum candidum, with the strain number MY-0603, which is deposited in the China Center for Type Culture Collection, and the deposit number is: CCTCC NO: M 2025455.

[0008] Strain name: Geotrichum candidum MY-0603;

[0009] The sample to be separated is enriched and cultured, transferred to a medium with polyethylene as the sole carbon source for enrichment screening and separation, and then a degrading bacterium Geotrichum candidum MY-0603 capable of using polyethylene as a carbon source and energy source is obtained.

[0010] The sample to be separated is a plastic sample in sewage.

[0011] The components of the medium with polyethylene as the sole carbon source are: NaNO 3 2 g, MgSO 4 0.5 g, K 2 HPO 4 1 g, FeSO 4 0.01 g, distilled water 1000 mL, KCL 0.5 g, pH: natural condition, which is a liquid medium; adding 15 - 20 g of agar above is a solid medium.

[0012] The colony morphological description characteristics of this strain are: the colony is white, circular in shape, with neat edges and clear outlines. The ITS of the strain was amplified and sequenced, and the obtained sequence was compared with the blastn (the latest version v2.13) nucleic acid database. Among them, the nucleic acid database selects the latest version of the nt library. By comparing with the nt library through blastn, the results show that the e value of the blast comparison result of MY-0603 and Geotrichum candidum is 0.0, and the homology with the reference species Geotrichum candidum reaches 98.93%. Combining the physiological and biochemical characteristics of the strain, MY-0603 was initially identified as Geotrichum candidum.

[0013] The present invention also provides the application of the above Geotrichum candidum in degrading polyethylene plastic mulch.

[0014] The Geotrichum candidum MY-0603 provided by the present invention has a good effect on degrading polyethylene plastic mulch film. After 21 days of biodegradation treatment, the surface of the polyethylene plastic mulch film is rough, with obvious cracks, pits and gullies on the surface, and the weight loss rate of the polyethylene plastic mulch film reaches 8%, providing a new strain resource for the biodegradation of polyethylene plastic mulch film, and having broad application prospects.

[0015] The present invention also provides the application of the polyethylene plastic mulch film degrading bacteria in the preparation of a polyethylene plastic mulch film degrading agent.

[0016] The present invention also provides a polyethylene plastic mulch film degrading agent, and the active ingredient contains the Geotrichum candidum described above.

[0017] The present invention also provides a preparation method of the polyethylene plastic mulch film degrading agent. The Geotrichum candidum is inoculated into a culture medium, and after cultivation, it is prepared into a bacterial liquid, which is the polyethylene plastic mulch film degrading agent.

[0018] Preferably, the cultivation temperature is 28 °C, and it is cultivated at 180 rpm for 24 h.

[0019] Preferably, the volume ratio of the Geotrichum candidum contained in the bacterial liquid is 10%.

[0020] The present invention also provides a method for degrading polyethylene plastic mulch film, using the Geotrichum candidum or the polyethylene plastic mulch film degrading agent described above to degrade the polyethylene plastic mulch film.

[0021] Specifically, the specific steps of using the Geotrichum candidum to degrade the polyethylene plastic mulch film are as follows:

[0022] (1) Cut the polyethylene plastic mulch film into small film pieces;

[0023] (2) Put the small film pieces into a sodium dodecyl sulfate solution to completely immerse them, take them out and ultrasonically clean them with 50% ethanol, then sequentially soak them in 10%, 30%, 50%, 75%, 90% and 100% ethanol, and then sterilize the small film pieces by ultraviolet for 30 min and put them into an oven to dry, obtaining sterilized small film pieces.

[0024] (3) Add the sterilized small film pieces into an inorganic salt culture medium, prepare the Geotrichum candidum strain into a bacterial liquid, and inoculate it into the inorganic salt culture medium with polyethylene plastic mulch film as the sole carbon source, and culture it by shaking flask oscillation.

[0025] Specifically, the Geotrichum candidum strain is inoculated at a volume ratio of 10% to prepare a bacterial liquid; the shaking flask oscillation culture conditions are 28 °C and 180 rpm.

[0026] The beneficial effects of the present invention:

[0027] By using the method for isolating and screening plastic-degrading bacteria of the present invention, strain resources capable of degrading polyethylene can be obtained. The strains obtained by the present invention have good effects on the biodegradation of polyethylene plastics. After 21 days of biodegradation treatment, the weight loss rate of polyethylene plastic mulch reaches 8%. The method of the present invention is green and environmentally friendly, low in cost, and convenient to operate. The obtained strains have good degradation characteristics, providing new resources and ideas for the bioremediation of polyethylene waste in the environment, and having broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 FIG. 6 is a diagram showing the colony morphological characteristics of polyethylene-degrading bacterium MY-0603 provided by the present invention;

[0029] Figure 2 FIG. 10 is a scanning electron microscope observation diagram of the colony morphology of polyethylene-degrading bacterium MY-0603 provided by the present invention;

[0030] Figure 3 FIG. 14 is a schematic diagram showing the growth curve of degrading bacterium MY-0603 provided by the present invention;

[0031] Figure 4 FIG. 18 is a scanning electron microscope characteristic diagram of the PE film after 21 days of degradation in the experimental group inoculated with degrading bacterium MY-0603 provided by the present invention;

[0032] Figure 5 FIG. 22 is a scanning electron microscope characteristic diagram of the PE film after 21 days of degradation in the experimental group not inoculated with degrading bacterium MY-0603 provided by the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0033] This strain has good degradation effects on plastic agricultural films, especially on polyethylene films. This method is green and environmentally friendly, low in cost, and convenient to operate. By adopting this biodegradation method, the degradation effect reaches 8±0.5% after 21 days of treatment, providing new resources and ideas for the bioremediation of polyethylene plastic films, and having broad application prospects.

[0034] Example 1

[0035] Isolation, screening and identification of polyethylene-degrading bacteria:

[0036] (1) Sampling time: Plastics in the sewage collected from the Donghu Campus of Zhejiang A&F University in Hangzhou, Zhejiang Province were used as samples.

[0037] (2) Use an inoculation loop to transfer the colonies on the surface of plastics in the sewage to a sterilized liquid medium with a PE film as the sole carbon source. Add a transparant plastic film sterilized by ultraviolet light, and culture at 28 °C for 3 days. Regularly select the fast-growing bacteria and repeat the culture 3 times. At the same time, set up 3 100 mL inorganic salt media, each inoculated with only one sterile membrane as a blank control group. By comparing with the blank control group, the experimental group's culture medium was significantly turbid, and the growth of strains in the culture medium could also be indicated by measuring OD600.

[0038] (3) Select the fast-growing culture system and gradually dilute it to prepare bacterial suspensions with different dilution degrees. Use the dilution plating method to isolate the strains, and repeatedly streak the isolated strains for purification.

[0039] (4) After obvious colonies grow on the plate, observe the size, color, and morphology of the colonies. Select different strains that can grow well with a PE film as the sole carbon source, mark the strains with different morphologies, and streak them again on different inorganic salt solid media. Culture in an incubator at 30 °C for 48 h. Streak the selected strains on the plate more than 3 times to purify the strains and obtain single colonies. Then streak the single colonies on a PDA solid medium and store them numbered in a refrigerator at 4 °C.

[0040] The culture medium for separation and screening is a carbon-free mineral medium, and its components are: NaNO 3 2 g, MgSO 4 0.5 g, K 2 HPO 4 1 g, FeSO 4 0.01 g, 1000 mL of distilled water, 0.5 g of KCL, pH: natural condition, which is the liquid medium; adding 15 - 20 g of agar above is the solid medium.

[0041] Through the above separation and screening work, after multiple separation and purification, a fast-growing polyethylene-degrading bacterium MY-0603 was obtained. This degrading bacterium shows a white filamentous shape on the inorganic salt culture.

[0042] The colony morphological characteristic diagram is as Figure 1 shown. The colony is white, circular in shape, with a neat edge and clear outline; the scanning electron microscope observation diagram of the colony morphology is as Figure 2 shown.

[0043] Physiological characteristics and molecular biological identification of the strain:

[0044] Extract DNA using a fungal genomic DNA extraction kit, amplify it with the universal primers ITS1 and ITS4 for strain identification. Analyze whether the PCR product bands are consistent with the target size, whether they are single, and whether there is smearing through agarose gel electrophoresis.

[0045] ITS sequence primers ITS1: 5'-TCCGTAGGTGAACCTGCGG-3′, ITS4: 5'-TCCTCCGCTTATTGATATGC-3′

[0046] PCR product sequencing: After the PCR product is qualified, the target band is cut for purification and recovery, and the recovered product is used for Sanger sequencing.

[0047] Sequencing result alignment and analysis:

[0048] 1. The Sanger sequencing results are spliced using the software ContigExpress, and the inaccurate parts at both ends are removed. The spliced sequence is shown as SEQ ID No.1.

[0049] 2. Batch blastn alignment of the spliced sequences against the nucleic acid database. Among them, the nucleic acid database selects the latest version of the nt library. Through sequence analysis, it is determined that the genus of this strain is Geotrichum, indicating that the isolated MY-63 strain is Geotrichum candidum. The newly screened strain is named Geotrichum candidum, strain number MY-0603, and it was deposited in the China Center for Type Culture Collection located at Wuhan University, China on March 12, 2025, with the deposit number CCTCC NO: M 2025455.

[0050] Example 2 Determination of the growth curve of strain MY-0603

[0051] The experimental materials used are PE films, and the plastic-degrading bacterium is strain MY-0603. Using the liquid shaking flask culture method, the growth curves of strain MY-0603 for 24 hours are measured respectively.

[0052] Specific operation: Mark the corresponding strain in the conical flask containing 100 mL of sterilized PDA medium in advance. Scrape the pure strain on the PDA plate and inoculate it into the sterilized PDA culture solution for 24-hour culture. Place the inoculated conical flask on a shaker and shake it at 37°C and 180 rpm. Take 4 mL of the bacterial solution from the conical flask every 2 hours, centrifuge it, and add sterile normal saline after centrifugation. Use the uninoculated sterile normal saline as the blank control group, and select a wavelength of 600 nm for turbidimetric determination by photoelectricity. Finally, draw the growth curve of the corresponding strain, as Figure 3 shown.

[0053] Example 3

[0054] 1. Experiment on the determination of the weight loss rate of PE film degraded by strain MY-0603

[0055] Sterile treatment: The PE film was first soaked in a 2% sodium dodecyl sulfate (SDS) solution for 4 h, then placed in 50% ethanol and ultrasonically cleaned for 30 min, twice in total. Then it was soaked in 70% ethanol overnight, and finally rinsed with absolute ethanol multiple times to ensure that the biofilm, impurities, etc. on the PE film were cleaned. Finally, the PE film was put into an oven and dried at a low temperature of 40 °C.

[0056] Cultivation experiment: The small PE film pieces of 5.0×5.0 cm were numbered and accurately weighed for the initial weight. After sterilization, they were added to the basic inorganic salt liquid medium, and the liquid loading amount was 100 mL / 250 mL conical flask. The MY-0603 strain was inoculated with the pre-prepared bacterial suspension at a volume ratio of 10%, and inoculated into 100 mL of liquid inorganic salt medium with the corresponding PE film as the sole carbon source. Three parallels were set for each strain as the experimental group. They were cultured by shaking flask at 180 rpm and 28 °C for 21 days. The PE film-inorganic salt liquid medium without inoculating the bacterial suspension was cultured under the same conditions as the blank control group.

[0057] Calculation of the weight loss rate of the PE film: The weight loss rate of different PE films after degradation by different test strains was calculated by the weighing method. After 21 days of culture, the small PE film pieces were taken out for sterile treatment, and weighed using a one-hundred-thousandth balance. The degradation strains were screened by calculating the degradation rate of the PE film, and the degradation effect of different plastics was characterized by the percentage loss before and after plastic degradation. The weight loss rate of the PE film = (initial weight of the PE film - weight of the PE film after degradation) / initial weight of the PE film.

[0058] Experimental results: After 21 days of culture, the weight loss rate results of the PE films in the experimental group (three parallels were experimental groups 1-3 respectively) and the blank control group are shown in Table 1. The average weight loss rate of the three experimental groups was 8.20%. It can be seen that the weight loss rate of using the MY-63 strain to degrade polyethylene plastic mulch can reach at least 8%, showing a good degradation effect.

[0059] Table 1 Weight loss rate of PE film after 21 days of culture

[0060]

[0061] 2. Scanning electron microscope observation experiment of the degradation of PE film by MY-0603 strain

[0062] Sterile treatment: The PE film was first soaked in a 2% sodium dodecyl sulfate (SDS) solution for 4 h, then placed in 50% ethanol and ultrasonically cleaned for 30 min, twice in total. Then it was soaked in 70% ethanol overnight, and finally rinsed with absolute ethanol multiple times to ensure that the biofilm, impurities, etc. on the PE film were cleaned. Finally, the PE film was placed in an oven and dried at a low temperature of 40 °C.

[0063] Cultivation experiment: The small PE film pieces of 5.0×5.0 cm were numbered and the initial weight was accurately weighed. After sterilization, they were added to the basic inorganic salt liquid medium, and the liquid loading volume was 100 mL / 250 mL Erlenmeyer flask. The strain MY-0603 was inoculated with the pre-prepared bacterial suspension according to a volume ratio of 10% into 100 mL of liquid inorganic salt medium with the corresponding PE film as the sole carbon source. Three parallels were set for each group of strains as the experimental group. It was cultured by shaking flask at 180 rpm and 30 °C for 21 days. The PE film-inorganic salt liquid medium without inoculating the bacterial suspension was cultured under the same conditions as the blank control group.

[0064] Scanning electron microscope observation: After taking out the small PE film pieces cultured for 21 days and removing the impurities on the surface of the PE film, a small piece of the film was carefully cut with scissors and stuck on the sample stage with conductive colloid. After fixing and spraying gold for 120 s, it was taken out and sent into the sample chamber. The vacuum switch was turned on, and the vacuum pump started to work, and the air in the electron chamber and the sample chamber was pumped out. When it became a completely vacuum state, the light outside the sample chamber would show blue. Then the switch of the SEM software on the computer was turned on. After determining the working voltage and working distance according to the conductivity of the sample and the height of the sample stage, the observation began. First, find the sample stage, determine the center, and gradually increase the magnification from 500 times. At the same time, adjust the clarity of the picture. After finding the wrinkles and cracks of the PE film, save the picture.

[0065] Experimental results: After 21 days of degradation in the experimental group inoculated with the degrading bacterium MY-0603, the PE film was as Figure 4 shown, with a rough surface and obvious cracks and pits on the surface. After 21 days of degradation in the blank control group without inoculating the degrading bacterium MY-0603, the PE film was as Figure 5 shown, with a smooth and flat surface. The experimental results showed that in the experimental group inoculated with the degrading bacterium MY-0603, the PE film underwent biodegradation.

Claims

1. A Geotrichum candidum, characterized in that The strain was named Geotrichum candidum, with the strain number MY-0603, and was deposited in China Center for Type Culture Collection with the deposit number: CCTCC NO: M 2025455.

2. Use of Geotrichum candidum as claimed in claim 1 in degrading polyethylene plastic mulch.

3. Use of Geotrichum candidum as claimed in claim 1 in preparing a degrading agent for degrading polyethylene plastic mulch.

4. A polyethylene plastic mulch film degradation agent, characterized in that: The active ingredient comprises the Geotrichum candidum described in claim 1.

5. The method for preparing the polyethylene plastic mulch film degradation agent according to claim 4, characterized in that: The Geotrichum candidum described in claim 1 is inoculated into a culture medium and cultured to prepare a bacterial liquid, which is a polyethylene plastic mulch film degrading agent.

6. The preparation method according to claim 5, characterized in that: The culture temperature was 28°C and the culture speed was 180 rpm for 24 h.

7. The preparation method according to claim 5, characterized in that: The volume ratio of the Geotrichum candidum in the bacterial liquid is 10%.

8. A method for degrading polyethylene plastic mulch, characterized in that: The polyethylene plastic mulch film is degraded by using the Geotrichum candidum described in claim 1 or the polyethylene plastic mulch film degrading agent described in claim 4.

9. The method for degrading polyethylene plastic mulch as claimed in claim 8, characterized in that: The specific steps of using the Geotrichum candidum described in claim 1 to degrade polyethylene plastic mulch: (1) Cut the polyethylene plastic mulch into small pieces; (2) The small membrane piece is placed in a sodium dodecyl sulfate solution to completely immerse it, and then it is taken out and ultrasonically cleaned with 50%, and then it is immersed in 10%, 30%, 50%, 75%, 90% and 100% ethanol in sequence, and then the small membrane piece is ultraviolet sterilized for 30 minutes and then placed in an oven for drying to obtain a sterilized small membrane piece. (3) Add the sterilized small film pieces to an inorganic salt culture medium, prepare a bacterial solution of the Geotrichum candidum strain, inoculate it into an inorganic salt culture medium with polyethylene plastic mulch as the sole carbon source, and culture it in a shake flask.

10. The method for degrading polyethylene plastic mulch according to claim 9, characterized in that: The Geotrichum candidum strain was inoculated at a volume ratio of 10% to prepare a bacterial solution; The shaking conditions of the shake flask culture were 28°C and 180 rpm.

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