Stenotrophomonas for degrading polyethylene plastic as well as application and preparation of stenotrophomonas

Abstract

The invention belongs to the technical field of agricultural microorganisms, and particularly relates to stenotrophomonas for degrading polyethylene plastics as well as application and a preparation of the stenotrophomonas. The stenotrophomonas is preserved in the China General Microbiological Culture Collection Center (CGMCC), and the preservation number is CGMCC No.32667. The nucleotide sequence of 16S rDNA of the strain is shown as SEQ ID NO: 1, and verification proves that the strain can secrete lipase and can biodegrade polyethylene plastics which are difficult to degrade in nature. According to the invention, a new biological resource is provided for biodegradation of polyethylene, and a new thought is provided for treatment of polyethylene solid waste.

Description

Technical Field

[0001] The invention belongs to the technical field of agricultural microorganisms and relates to an oligotrophic monocytogenes capable of degrading polyethylene plastics and an application and a preparation thereof. Background Art

[0002] Polyethylene (PE) is a long carbon chain polymerized from ethylene monomers. It has excellent chemical resistance, electrical insulation, low temperature resistance, and strong resistance to biodegradation. PE, as an inert material that is extremely difficult to degrade, was buried in moist soil and showed only partial degradation and negligible weight loss after 32 years. As early as 1974, researchers began to isolate microorganisms, hoping to use the characteristics of microorganisms to degrade PE. After decades of research, although researchers have found some microorganisms that can degrade PE from insects, soil, landfills and oceans, it is far from enough for the huge amount of PE waste plastics. It is still necessary to obtain more microorganisms that can be used for PE degradation through screening to provide new biological resources for PE biodegradation. Summary of the invention

[0003] In order to solve the above technical problems, the present invention isolates and screens a strain of Stenotrophomonas sp.YLE11 from a soil sample of a garbage dump in Yangling, Shaanxi Province. In-depth research on the strain found that the Stenotrophomonas sp.YLE11 can biodegrade PE.

[0004] In one aspect, the present invention provides a Stenotrophomonas sp., named Stenotrophomonas sp. YLE11.

[0005] Specifically, the Stenotrophomonas sp.YLE11 was screened and isolated from a soil sample from a garbage dump in Yangling, Shaanxi Province. The deposit information of the Stenotrophomonas sp.YLE11 is as follows:

[0006] Strain name: YLE11;

[0007] Classification name: Stenotrophomonas sp.;

[0008] Date of receipt by the depository: November 18, 2024;

[0009] Time of issuance of the preservation certificate: November 25, 2024;

[0010] Depository: China General Microbiology Center (CGMCC);

[0011] Deposit number: CGMCC No.32667.

[0012] Address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0013] Furthermore, the nucleotide sequence of 16S rDNA of Stenotrophomonas sp. YLE11 is shown in SEQ ID NO: 1.

[0014] Furthermore, the Stenotrophomonas sp. YLE11 can secrete lipase.

[0015] Furthermore, the Stenotrophomonas sp. YLE11 can secrete lipase.

[0016] Furthermore, the culture temperature of the Stenotrophomonas sp.YLE11 is 20°C-35°C.

[0017] Preferably, the culture temperature of Stenotrophomonas sp. YLE11 is 25°C.

[0018] On the other hand, the present invention provides the use of the oligotrophic mononas in degrading polyethylene plastics.

[0019] Furthermore, the oligotrophic mononas is applied to the biodegradation of polyethylene film, polyethylene particles or polyethylene powder.

[0020] In addition, the present invention also provides a preparation containing the fermentation broth of the oligotrophomonas.

[0021] It is easy for a person skilled in the art to know that in order to give full play to the efficacy of Stenotrophomonas sp. YLE11, it can be prepared into a preparation form that is easy to use according to the needs of use. Based on this, further, the present invention provides a preparation containing a fermentation broth of Stenotrophomonas sp. YLE11. The preparation is processed into an industrially accepted preparation form, and the preparation contains an auxiliary agent that assists Stenotrophomonas sp. YLE11 in improving the degradation of polyethylene plastic. The preparation can be formulated into a soluble liquid, water-dispersible granules, wettable powder, dispersible oil suspension, suspension, microemulsion, granule. When preparing the above-mentioned different dosage forms, for a person skilled in the art, in addition to using a fermentation broth containing the optional Stenotrophomonas sp. YLE11, it is also necessary to select a variety of auxiliary agents, and different preparation auxiliary ingredients (auxiliaries) can be selected and used as needed. The auxiliary component may be one or more of a dispersion medium, a dispersant, an emulsifier, a wetting agent, a thickener, a defoaming agent, an antifreeze agent, a disintegrant, a binder, a filler, a carrier, and the like.

[0022] In addition, the present invention also provides the application of a preparation containing the fermentation broth of the oligotrophomonas in biodegradable polyethylene plastics.

[0023] Furthermore, the preparation containing the oligotrophic mononas is applied to the biodegradation of polyethylene film, polyethylene particles or polyethylene powder.

[0024] Compared with the prior art, the technical solution provided by the present invention has at least the following beneficial effects or advantages:

[0025] The present invention screens and separates a strain of Stenotrophomonas sp.YLE11 from a garbage dump in Yangling, Shaanxi Province, and the strain has a good degradation effect on polyethylene plastics. The strain of the present invention is used to degrade polyethylene film, and after 31 days, the surface of the polyethylene film is rough, uneven, has holes, and is seriously damaged; the Stenotrophomonas sp.YLE11 provided by the present invention can biodegrade polyethylene plastics, and is applied to the biodegradation of polyethylene plastics, which has the advantages of being green, environmentally friendly, low cost, and easy to operate. The present invention provides a new biological resource for the treatment of polyethylene waste in the environment. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1The results of the extracellular lipase activity assay of Stenotrophomonas sp.YLE11; (a) Escherichia coli, a negative control, did not produce orange fluorescent substance on the Rhodamine B-olive oil medium; (b) strain YLE11, which produced orange fluorescent substance on the Rhodamine B-olive oil medium.

[0027] Figure 2 This is a diagram of the colony morphology characteristics of Stenotrophomonas sp.YLE11.

[0028] Figure 3 This is the morphological image of Stenotrophomonas sp.YLE11 under a scanning electron microscope.

[0029] Figure 4 (a) is the Gram staining image of Stenotrophomonas sp.YLE11; (b) is the Gram staining image of Escherichia coli G - ; (b) is strain YLE11; (c) is Staphylococcus aureus G + .

[0030] Figure 5 This is the result of PCR amplification and electrophoresis of the 16S rRNA gene of Stenotrophomonas sp.YLE11.

[0031] Figure 6 This is the phylogenetic tree of Stenotrophomonas sp.YLE11.

[0032] Figure 7 This is the growth curve of Stenotrophomonas sp.YLE11 at different temperatures.

[0033] Figure 8 The surface morphology of PE plastic film under scanning electron microscope; (a) is untreated PE, original plastic film; (b) is control: PE is degraded by no inoculated strain; (c) is E.coli: PE is degraded by inoculated wild-type E.coli; (d) is YLE11: PE is degraded by inoculated Stenotrophomonas sp.YLE11 strain.

[0034] Fig. 9 The surface morphology of PE plastic film under atomic force microscopy. Untreated PE: original plastic film; Control: PE degraded by no inoculated strain; E.coli: PE degraded by inoculated wild-type E.coli; YLE11: PE degraded by inoculated Stenotrophomonas sp.YLE11 strain.

[0035] Fig.10 The FTIR spectrum of PE plastic film. Untreated PE: original plastic film; Control: PE degraded by no inoculated strain; E.coli: PE degraded by inoculated wild-type E.coli; YLE11: PE degraded by inoculated Stenotrophomonas sp.YLE11 strain.

[0036] Fig.11 It is the characteristic absorption peak of -OH of PE plastic film. Untreated PE: original plastic film; Control: PE degraded by no inoculated strain; E.coli: PE degraded by inoculated wild-type E.coli; YLE11: PE degraded by inoculated Stenotrophomonas sp.YLE11 strain. DETAILED DESCRIPTION

[0037] The technical scheme of the present invention is described below in conjunction with the embodiments, but the present invention is not limited to the following embodiments. The experimental methods and detection methods described in each embodiment are conventional methods unless otherwise specified; the reagents and materials described are all commercially available unless otherwise specified.

[0038] Example 1

[0039] This example is the screening and separation of Stenotrophomonas sp.YLE11.

[0040] Test soil samples: Soil was collected from the soil near the aged plastic bags in the Yangling garbage dump in Shaanxi Province and randomly sampled and mixed, then transported to the laboratory at 4°C for storage.

[0041] Test plastics: polyethylene (PE) film, size 1×1 cm; polyethylene powder, powder particle size 80-200 μm.

[0042] Weigh 10.0 g of the freshly collected test soil sample, transfer it to a 250 mL conical flask containing 100 mL of sterile water, shake it at room temperature at 180 rpm for 30 min, and take the supernatant as the soil microbial inoculum after standing. Prepare 100 mL of inorganic salt liquid culture medium for each 250 mL conical flask and sterilize it at 121°C for 30 min. Inoculate the treated sterile plastic film or powder into the above culture medium at an inoculum amount of 0.5 wt% (0.5 g) to prepare a solid culture medium with polyethylene plastic as the sole carbon source. Prepare LB solid culture medium, sterilize it at 121°C for 30 min, and then pour it into a plate for use. Perform gradient dilution of the soil microbial inoculum, and take 10 -350 μL of the concentration dilution was spread on the LB solid medium plate, and each treatment was repeated 3 times. After spreading, the plate was inverted and cultured at 28°C in the dark for 2-3 days, and the bacterial species were classified and counted according to the colony morphology. The strains were then purified by streaking until a purified single colony appeared on the plate, and the pure strains were stored in a slant tube and placed in a 4°C refrigerator for use. A total of 220 strains of PE microplastic-degrading bacteria were isolated and purified using the sole carbon source method, of which 30 strains were YLE according to morphological classification.

[0043] Example 2

[0044] This example is a test for the extracellular lipase activity of the LYE strain.

[0045] Rhodamine B-olive oil agar solid culture medium: 10 g of peptone, 5 g of yeast extract, 10 g of sodium chloride, and 15 g of agar are diluted to 1000 mL and sterilized. When the temperature reaches 45°C, 31.25 mL of sterile filtered olive oil and 2.5 mL of Rhodamine B mother solution (mother solution concentration 4 mg / mL) are added in sequence, and the plates are poured to obtain Rhodamine B-olive oil agar solid culture medium.

[0046] Single clones of strains YLE1-YLE30 were selected and spread on rhodamine B-olive oil agar plates, and then cultured at 30°C for 2-3 days to observe whether orange fluorescent substances were produced. Escherichia coli (E. coli) was used as a negative control. E. coli was spread on rhodamine B-olive oil agar plates, and then cultured at 30°C for 2-3 days to observe whether orange fluorescent substances were produced. The test was considered valid if the E. coli group did not produce orange fluorescent substances.

[0047] Lipase can hydrolyze olive oil to produce fatty acids, and fatty acids react with the cation of Rhodamine-B to produce orange fluorescent substances on the plate. Lipase is believed to be related to plastic degradation, so lipase activity determination can be used as a preliminary screening of potential plastic-degrading bacteria. If the test strain produces orange fluorescent substances, it indicates that the strain can secrete lipase.

[0048] The results of the extracellular lipase activity test of LYE strain are as follows Figure 1 As shown, the negative control Escherichia coli (E. coli) group did not produce orange fluorescent substances, indicating that this test was effective. Stenotrophomonas sp. YLE11 produced orange fluorescent substances, indicating that it can produce lipase and has potential plastic degradation properties.

[0049] Example 3

[0050] This example is the morphological and biochemical identification of Stenotrophomonas sp.YLE11.

[0051] Pick a single colony of Stenotrophomonas sp.YLE11 and streak it on LB solid medium for 2 days. The results are as follows: Figure 2 As shown, the results showed that the colony size of Stenotrophomonas sp.YLE11 was smaller.

[0052] The morphology of Stenotrophomonas sp.YLE11 was observed by scanning electron microscopy (SEM). First, the Stenotrophomonas sp.YLE11 bacteria were fixed. The specific operation steps were as follows: (1) The bacterial suspension was inoculated into 100 mL of liquid LB culture medium at a volume percentage of 2% (v / v). After culturing for 20 hours, the above culture solution was centrifuged at 8000 rpm for 3-5 minutes, the supernatant was discarded, and centrifuged again until a mung bean-sized bacterial precipitate appeared at the bottom of the centrifuge tube after multiple centrifugations. The bacterial precipitate was taken for fixation. (2) 2 mL of 2.5% (v / v) glutaraldehyde was used for each sample to fully suspend the bacteria collected in the above steps, and the cells were fixed for 4 hours. (3) The above fixed bacteria were washed 2-3 times with 2 mL of PBS buffer solution with a pH of 7.2 and a concentration of 0.1 M, each time for 10 minutes. Then, the fixed cells were washed with 30%, 50%, 70%, 80%, and 90% ethanol for 10 minutes each time, and then washed twice with 100% ethanol for 10 minutes each time. Figure 3 As shown, the Stenotrophomonas sp.YLE11 is rod-shaped under a scanning electron microscope.

[0053] Gram staining of Stenotrophomonas sp.YLE11 was performed, with Escherichia coli (a) and Staphylococcus aureus (c) as controls. The results are shown in Figure 4 As shown, Stenotrophomonas sp.YLE11 is a Gram-negative bacterium.

[0054] Example 4

[0055] This example is the molecular identification of Stenotrophomonas sp.YLE11.

[0056] A single colony of purified Stenotrophomonas sp.YLE11 was picked and made into a bacterial suspension, and then 16S rDNA was amplified using bacterial universal primers. The information of bacterial universal primers is as follows:

[0057] 27F: 5'-AGAGTTTGATCCTGGCTCAG-3',

[0058] 1492R: 5'-GGTTACCTTGTTACGACTT-3'.

[0059] Results of PCR amplification and electrophoresis of 16S rRNA gene of Stenotrophomonas sp.YLE11 strain Figure 5 As shown, lane M is a marker, and lane 1 is Stenotrophomonas sp.YLE11. The size of the amplified product of the 16S rRNA gene of Stenotrophomonas sp.YLE11 in lane 1 is in the range of 1000bp-2000bp, indicating that the 16S rRNA gene was successfully amplified from the single clone of the strain Stenotrophomonas sp.YLE11. The single clone PCR amplified product of the strain Stenotrophomonas sp.YLE11 was sent to a biological company for sequencing, and the sequencing result is shown in SEQ ID NO: 1.

[0060] The sequencing results were copied to the NCBI database, and the 16SrDNA gene sequences of multiple related strains were obtained by BLAST comparison. MEGAX software was used for multiple sequence alignment and sorted according to the strain name. A phylogenetic tree was constructed based on the NJ method to determine the taxonomic status of the strain. Figure 6 The results showed that Stenotrophomonas sp.YLE11 belonged to the genus Stenotrophomonas.

[0061] Example 5

[0062] This example is a test on the most suitable growth temperature of Stenotrophomonas sp.YLE11.

[0063] Inoculate a single clone of Stenotrophomonas sp.YLE11 into 100 mL of liquid LB medium and culture overnight until OD 600 The above overnight cultured Stenotrophomonas sp.YLE11 bacterial solution was transferred to a 250mL conical flask containing 100mL liquid LB medium at a 1% (v / v) inoculation volume and cultured in a shaker at 200rpm in the dark at different temperatures, and the culture temperatures were 20, 25, 30, 35, 40, and 50°C. Samples were taken at 0, 2, 4, 6, 12, 18, and 24h of culture time, and the bacterial OD was measured. 600The effect of different temperatures on the growth of Stenotrophomonas sp.YLE11 was evaluated. The test results are shown in Figure 7 As shown, Stenotrophomonas sp.YLE11 can grow and reproduce in the range of 20℃-40℃, grow well in the range of 20℃-35℃, and the optimal growth temperature is 25℃.

[0064] Example 6

[0065] This example is a test on the degradation of polyethylene by Stenotrophomonas sp. YLE11.

[0066] The experiment set up Stenotrophomonas sp.YLE11 (experimental group), bacteria E. coli (negative control group) and sterile water group (blank control). The strains of different experimental groups and sterile water were pre-cultured, and then inoculated into inorganic salt culture medium containing PE film for 31 days. The changes of PE surface before and after different treatments were tested to evaluate the biodegradation effect of different treatments on PE.

[0067] Field emission electron microscopy (SEM) was used to evaluate the changes in the surface morphology of PE films before and after degradation, including surface degradation, hole formation, and cracks, in order to evaluate the effects of different treatments on PE biodegradation. Figure 8 As shown in the figure, the surface of untreated PE is smooth, and there is almost no change in PE before and after treatment with the blank control and negative control (E. coli), and the surface of PE is smooth. However, in the group treated with Stenotrophomonas sp.YLE11, the surface of PE membrane became rough and uneven after 31 days, and obvious holes appeared on the surface. These changes in surface morphology indicate that Stenotrophomonas sp.YLE11 has a significant degradation effect on PE membrane.

[0068] Atomic force microscopy (AFM) was used to visually display the surface roughness of PE membranes and verify the biodegradability of PE. Fig. 9As shown, the surface of the control PE membrane without bacterial culture inoculation is smooth, the surface of the negative control PE membrane inoculated with wild-type Escherichia coli E. coli has some protrusions, and the surface of the PE membrane inoculated with Stenotrophomonas sp. YLE11 is rougher than the blank control and the negative control. The surface roughness of the group treated with Stenotrophomonas sp. YLE11 varies greatly, and the uneven surface morphology is more conducive to the attachment of microorganisms and is conducive to further degradation, indicating that Stenotrophomonas sp. YLE11 has a significant degradation effect on PE.

[0069] NanoScope Analysis 3.00 software was used to reveal the changes in the roughness of PE diaphragms after different treatments. The results are shown in Table 1. Table 1 shows the surface roughness coefficients Rq, Ra and Rmax of the samples in each treatment group and control group, which respectively represent the root mean square of the surface roughness, the average value of the surface roughness, and the maximum peak-to-valley height. As can be seen from Table 1, the roughness parameter values ​​of the control group and the negative control group are low, indicating that the surfaces of the blank control group and the negative control group are relatively smooth. The surface roughness of the diaphragm in the group treated with Stenotrophomonas sp.YLE11 changed greatly, and the biodegradation effect on PE was stronger.

[0070] Table 1 Surface roughness parameters of PE membranes in different treatment groups (nm)

[0071] Treatment Group R Ra Rmax Blank control 8.81 7.05 49.1 E. coli 10.7 8.75 56.4 YLE11 43.6 34.5 290

[0072] Note: Different letters represent different treatment groups; Rq: root mean square roughness; Ra: average roughness; Rmax: maximum peak-to-valley height.

[0073] FTIR spectroscopy was used to analyze the changes in the chemical structure of PE before and after biodegradation. -1 It is -CH2 antisymmetric stretching vibration, 2850cm -1 They are -CH2 symmetric stretching vibration, 1470cm -1 They are -CH2 shear vibration, 1370cm -1 They are -CH3 symmetrical angle vibration, and 721cm -1 They are -CH2 plane swings. The results are as follows Fig.10 As shown in the figure, the infrared characteristic peaks corresponding to PE in all PE plastic sheets did not shift, but compared with the untreated PE, the control group, the negative control group, the infrared characteristic peaks corresponding to PE in the treated group Stenotrophomonas sp.YLE11 weakened, indicating that the functional groups of the PE film sheets changed. In addition, Fig.11As shown, wavelength 3389cm –1 The -OH characteristic absorption peak is shown in Figure 2. After biodegrading PE, the -OH absorption peak of the treated group Stenotrophomonas sp.YLE11 became wider and stronger compared with the control group, negative control group and untreated PE. This is related to the breaking of bonds in the PE molecules during the degradation process, which releases more hydroxyl groups, indicating that Stenotrophomonas sp.YLE11 can biodegrade PE.

[0074] In addition, the strain of the present invention or the bacterial agent containing the strain of the present invention can also degrade polyethylene plastic in the form of particles or powders. The polyethylene plastic particles or powders are directly added to a solid or liquid culture medium, and then the strain of the present invention is inoculated in the above culture medium for cultivation until the polyethylene plastic is completely degraded.

[0075] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A oligotrophic monocytogenes, characterized in that It was named Stenotrophomonas sp.YLE11 and deposited in the General Microbiology Center of China Culture Collection Administration with the deposit number CGMCC No.32667.

2. The oligotrophic mononas according to claim 1, characterized in that The nucleotide sequence of 16S rDNA of Stenotrophomonas sp. YLE11 is shown in SEQ ID NO:

1.

3. The oligotrophic mononas according to claim 1, characterized in that The Stenotrophomonas sp. YLE11 can secrete lipase.

4. The oligotrophic mononas according to claim 1, characterized in that The culture temperature of the Stenotrophomonas sp.YLE11 is 20°C-35°C.

5. The oligotrophic mononas according to claim 4, characterized in that The culture temperature of the Stenotrophomonas sp.YLE11 is 25°C.

6. Use of the oligotrophic mononas described in claim 1 in degrading polyethylene plastics.

7. The use according to claim 6, wherein the polyethylene plastic is polyethylene film, polyethylene particles or polyethylene powder.

8. A preparation containing the fermentation broth of the Stenotrophomonas according to claim 1.

9. Use of the preparation according to claim 8 in degrading polyethylene plastics.

10. The use according to claim 9, wherein the polyethylene plastic is polyethylene film, polyethylene particles or polyethylene powder.

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