Pseudomonas tingii SCSIO 85030 capable of efficiently degrading plastics and application of pseudomonas tingii SCSIO 85030

Abstract

The invention discloses a strain of Pseudomonas iranensis SCSIO 85030 and an application of the strain of Pseudomonas iranensis SCSIO 85030. The preservation number of the pseudomonas tingii SCSIO 85030 is GDMCC (China General Microbiological Culture Collection Center) No. 66488. The strain is preserved in Guangdong Microbial Culture Collection Center (GDMCC) on June 9, 2025, the preservation address is the 5th floor of the Experimental Building, No.100 Court, Middle Road, Xianlie, Guangzhou, Guangdong, and the postcode is 510070. The strain can generate various esterase such as extracellular urease, protease and the like, and when the strain is inoculated on an inorganic salt solid plate which takes waterborne polyurethane Impranil TMDLN and solid polyurethane PBA-PU as unique carbon sources, an obvious hydrolytic transparent ring can appear after 6 days. The Pseudomonas tingii SCSIO 81030 provided by the invention has the function of degrading polyurethane (PU) with different structural complexities, and can provide theoretical guidance and technical support for the recycling of the PU in the future.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and particularly relates to a strain of Pseudomonas sivasensis SCSIO 85030 capable of efficiently degrading plastics and an application thereof. Background Art

[0002] Plastic, a synthetic polymer, has become an absolute necessity in human production and life since its creation, bringing revolutionary changes to human life, the social economy, and industries such as agriculture, industry, and medicine. However, while the mass production of plastics has brought immense convenience to people's lives, it has also generated a significant amount of waste. According to statistics, as of 2015, approximately 8.3 billion tons of plastic were generated globally, of which 6.3 billion tons had become landfill waste. Only 9% of this was recycled, 12% was incinerated, and 79% remained in landfills or the natural environment. Plastic waste is widely distributed, found in terrestrial, marine, and even Antarctic ecosystems worldwide. Microplastic particles have been detected in plants, animals, and even humans, posing a serious threat to ecosystem balance and human health. Polyurethane (PU) is a polymer synthesized by the reaction of diisocyanates, short-chain diols (carbon chain extenders), and polyols. Its structure contains recurring urethane bonds (-NHCOO-). It is commonly used in the production of foams, elastomers, fiber plastics, fibers, leather and shoe resins, coatings, adhesives, and sealants. According to statistics, the output of PU has accounted for 7.9% of the world's total plastic production, ranking sixth in global plastic usage. The structure of PU contains amorphous soft segments and ordered hard segments. Among them, the hard segment is formed by the reaction of diisocyanate and short-chain diols, containing urethane bonds, while the soft part is composed of polyester or polyether polyols, providing elasticity and flexibility. The strong polyurethane bonds, unique microstructure and morphology in the PU structure make this type of plastic extremely durable and difficult to degrade naturally. At present, the main methods of disposing of a large amount of PU waste are landfill and incineration, which is not only costly and unsustainable, but also increases carbon dioxide emissions and releases a large amount of microplastics and toxic substances (such as aromatic diamine compounds) into the environment, seriously polluting the environment and threatening human health.

[0003] Biodegradation is an environmentally friendly plastic treatment method. It degrades high-molecular plastics into oligomers through the action of microorganisms, and finally mineralizes them into water and carbon dioxide. It has the advantages of being environmentally friendly and sustainable, and can effectively avoid the serious environmental pollution problems caused by traditional waste disposal methods. Although there are currently species from genera such as Pseudomonas, Bacillus, Comamonas, Aspergillus, Cladosporium and Penicillium that have been reported to be able to degrade PU, the number of these strains is small and the efficiency is generally low, which seriously limits the application of biodegradation in PU plastic treatment. This patent screens Impranil, a water-based polyester polyurethane that can simultaneously degrade TM The strains of DLN and solid polyester polyurethane - Poly(1,4-butylene adipate)-based PU (Poly(1,4-butylene adipate)-based PU, PBA-PU) were cultivated and applied to the degradation of PU waste in the natural environment, which has important application value for ecological environmental protection. Summary of the Invention

[0004] The present invention aims to solve the problem of low degradation efficiency of existing PU biodegradation strains and provide a PU-degrading strain Pseudomonas sivasensis SCSIO 85030. The strain can produce a variety of extracellular degradation enzymes, including urease and protease, and can simultaneously and efficiently degrade water-soluble polyester polyurethane Impranil. TM DLN and solid polyester polyurethane PBA-PU.

[0005] The first object of the present invention is to provide a strain of Pseudomonas sivasensis SCSIO 85030, with a deposit number of GDMCC No. 66488. It was deposited in the Guangdong Provincial Microbiological Culture Collection Center (GDMCC) on June 9, 2025, and the deposit address is: 5th Floor, Dayuan Experimental Building, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province, Postal Code: 510070.

[0006] The second object of the present invention is to provide a microbial preparation comprising the above-mentioned Pseudomonas sivasensis SCSIO 85030 as an active ingredient.

[0007] The third object of the present invention is to provide the use of the above-mentioned Pseudomonas sivasensis SCSIO 85030 or the above-mentioned preparation in degrading plastics.

[0008] Preferably, the plastic is water-soluble polyester polyurethane Impranil TM DLN and solid polyester polyurethane PBA-PU.

[0009] Specifically, the application is that Pseudomonas sivasensis SCSIO 85030 produces extracellular degradation enzymes to degrade polyurethane plastics.

[0010] Preferably, the degradation enzyme is protease or urease.

[0011] The present invention also provides the above-mentioned method for degrading plastics, which uses Pseudomonas syvassociens SCSIO 85030 to degrade plastics.

[0012] The Pseudomonas sivasensis SCSIO 85030 obtained by the present invention can produce urease and protease. In the urease activity test, it produces a distinct red color on a phenol red urea agar plate. In the protease activity test, it can liquefy gelatin in a culture tube.

[0013] Advantages of the present invention:

[0014] The present invention obtains Pseudomonas sivasensis SCSIO 85030, which can produce extracellular urease and protease, and inoculates the strain in an Impranil TM On inorganic salt solid plates with DLN and PBA-PU as the sole carbon source, an obvious hydrolysis transparent zone can be seen after 6 days. The above results show that Pseudomonas sivasensis SCSIO 85030 can hydrolyze two polyurethanes with different structural complexity (water-soluble polyurethane Impranil TM Both DLN and solid polyurethane (PBA-PU) showed good degradation properties.

[0015] Pseudomonas sivasensis SCSIO 85030, deposited with GDMCC No. 66488, was deposited on June 9, 2025, at the Guangdong Microbial Culture Collection Center (GDMCC), 5th Floor, Dayuan Experimental Building, 100 Xianlie Middle Road, Yuexiu District, Guangzhou, Guangdong, China, 510070, China. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is a colony morphology diagram of Pseudomonas sivasensis SCSIO 85030 on LB solid culture medium.

[0017] Figure 2 This is a microscopic structure diagram of Pseudomonas sivasensis SCSIO 85030.

[0018] Figure 3 and Figure 4 Pseudomonas sivasensis SCSIO 85030 and Impranil TM The effect of degrading PU to produce transparent circles in inorganic salt solid plates with DLN and PBA-PU as the only carbon sources (6th day after inoculation).

[0019] Figure 5 This is the urease detection test plate for Pseudomonas sivasensis SCSIO 85030.

[0020] Figure 6 The figure shows the gelatin liquefaction test results of Pseudomonas sivasensis SCSIO 85030. From left to right, the first tube is the control group, and tubes 3-4 are the experimental groups. DETAILED DESCRIPTION

[0021] The present invention is further explained below in conjunction with the accompanying drawings and specific examples, but does not constitute any limitation to the present invention. Unless otherwise specified, the following examples are all conventional reagents and method steps in the art.

[0022] Example 1: Isolation and preservation of strain SCSIO 85030

[0023] The strain 85030 described in the present invention was isolated from marine sediments in Daya Bay, Huizhou. The sample was collected on October 10, 2024, at 22°35.60'N, 114°33.70'E. In a clean bench, 1 g of sediment was weighed into a 250 mL sterile shake flask, 100 mL of sterile water was added, and after shaking, 200 μL of the suspension was drawn and applied to a 1% volume fraction of Impranil TM DLN was cultured in a oligotrophic MSM inorganic salt solid medium at a constant temperature for 1 month. A single colony with a transparent circle was picked with an inoculation loop and transferred to LB medium supplemented with 5% by volume and 1% by volume Impranil TMDLN was subcultured on MSM inorganic salt solid medium, its species was determined after purification and stored in 20% sterilized glycerol tubes, and the strain SCSIO85030 was screened.

[0024] MSM inorganic salt medium formula: Na2HPO4 2.8g / L, (NH4)2SO4 0.5g / L, CuCl2·2H2O0.001mg / L, H3BO3 0.03mg / L, FeSO4.·7H2O 0.2mg / L, MnCl 2· 4H2O 0.003mg / L, NiCl2·6H2O 0.002mg / L, KH2PO4 1g / L, Na2EDTA 0.5mg / L, CoCl2·6H2O 0.02mg / L, ZnSO4·7H2O 0.01mg / L, Na2MoO4·2H2O 0.003mg / L, solvent: water (pH 7.2). Solid culture medium is prepared by adding 15g / L agar. The preparation method is to mix all ingredients, adjust the pH, and sterilize.

[0025] LB medium formula: tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, distilled water 1000 mL, pH 7.2-7.4.

[0026] Example 2: Classification and Identification of Strain SCSIO 85030

[0027] (1) Example 1 Screening of strain 85030 Colony morphology: The colonies of this strain on LB medium are irregular circles. The colony color is white at the beginning of the culture, and becomes light yellow as the culture time increases ( Figure 1 The morphology of bacteria was observed under a scanning electron microscope at 5.0k× ( Figure 2 ).

[0028] (2) Molecular biological identification

[0029] DNA extraction: Strain 85030 was streaked onto LB medium plates and cultured at 28°C for 3 days. Purified single colonies were collected and transferred to 2 mL sterile EP tubes. DNA was extracted using the Chelx-100 method. Phylogenetic analysis was performed using rDNA-ITS gene sequences. PCR primers for rDNA-ITS gene amplification were synthesized by Shanghai Sangon Co., Ltd. Primer sequences were: Primer 1: 27F 5'-AGAGTTTGATCCTCGCTCAG-3'; Primer 2: 1492R 5'-TACGGCTA CCTTGTTACGACTT-3'. The PCR reaction system was as follows:

[0030]

[0031]

[0032] PCR amplification conditions: pre-denaturation at 95°C for 5 min; denaturation at 94°C for 30 s, annealing at 56°C for 30 s, extension at 72°C for 30 s, 35 cycles; final extension at 72°C for 5 min; storage at 4°C. PCR amplification products were detected by 1% agarose gel electrophoresis, and the PCR products were recovered and purified using an agarose gel DNA recovery kit (spin column type). After purification, the PCR product was sent to Guangzhou Tianyi Huiyuan Company for sequencing, and its sequence is shown in SEQ ID NO.1. Comparison of this sequence with known standard strains in the EzBioCloud database showed that this strain had the highest homology with Pseudomonas sivasensis, reaching 100%. Therefore, the strain was classified as belonging to the Bacteria kingdom, Pseudomonadota phylum, Gammaproteobacteria class, Pseudomonadales order, Pseudomonadaceae family, Pseudomonas genus, and Pseudomonas sivasensis (Pseudomonas sivasensis SCSIO 85030). The strain was named Pseudomonas sivasensis SCSIO 85030 and the deposit number was GDMCC No. 66488. It was deposited with the Guangdong Provincial Microbial Culture Collection (GDMCC) on June 9, 2025, at the 5th Floor, Dayuan Experimental Building, 100 Xianlie Middle Road, Yuexiu District, Guangzhou, Guangdong Province, Postal Code: 510070.

[0033] Example 3: Enzyme activity test of Pseudomonas sivasilis SCSIO 85030

[0034] In the urease activity test, Pseudomonas sylvasiae 85030 was inoculated on a urease phenol red urea solid culture medium plate and cultured at 28°C. The plate was observed every 24 hours to see if it turned from yellow to red. On the 4th day after inoculation, the center of the plate turned red ( Figure 5 ).

[0035] In the protease experiment, Pseudomonas sylvasiae 85030 was inoculated into gelatinase gelatin liquefaction solid slant medium, with 3 replicates (experimental group), and gelatinase gelatin liquefaction solid slant medium (no inoculation) was used as blank control (control group); cultured at 28°C, gelatin liquefaction was observed every 24 hours, and after 7 days of inoculation, it was found that the bacteria had obvious gelatin liquefaction ( Figure 6 ).

[0036] Urease phenol red urea solid culture medium formula: 1g / L peptone, 5g / L NaCl, 1g / L glucose, 2g / L KH2PO4, 0.012g / L phenol red, 15g / L agar, 40% urea solution, solvent is water (pH 6.8). Preparation method is to mix all ingredients, adjust pH, and sterilize.

[0037] The formula for the gelatinase gelatin liquefaction solid slant medium is: 5g / L peptone, 20g / L glucose, 200g / L gelatin, and the solvent is water (pH 7.4). The preparation method is to mix the ingredients uniformly, adjust the pH, and sterilize.

[0038] Example 4: Degradation of Impranil by Pseudomonas sivasilis SCSIO 85030 TM Detection of DLN

[0039] Preparation of waterborne polyurethane Impranil TM PU solid medium with DLN as the sole carbon source [volume fraction 0.3% polyurethane (PU), NaCl 2.2 g / L, Na2HPO4 2.8 g / L, (NH4)2SO4 0.5 g / L, CuCl2·2H2O 0.001 mg / L, H3BO3 0.03 mg / L, FeSO4.·7H2O 0.2 mg / L, MnCl 2· 4H2O 0.003mg / L, NiCl2·6H2O 0.002mg / L, KH2PO41g / L, Na2EDTA0.5mg / L, CoCl2·6H2O 0.02mg / L, ZnSO4·7H2O 0.01mg / L, Na2MoO4·2H2O0.003mg / L, agar 7.5g / L, solvent is water], of which PU (Impranil TM DLN) is a water-based polyurethane, a commercial anionic aliphatic colloidal polyester PU dispersion produced by Bayer, and is widely used in the textile and leather industries. Pseudomonas syvase 85030 was inoculated into a PU solid culture medium with water-based polyurethane as the sole carbon source. After inverted cultivation at 28°C in the dark for 6 days, the production of hydrolysis circles was observed. Figure 3 As shown, a transparent hydrolysis zone with a diameter of 14 mm was produced after 6 days of culture, which clearly showed the characteristics of producing extracellular hydrolases. The test results showed that the polyurethane in the culture medium was degraded and the DLN solid culture produced an obvious transparent zone on the 6th day of inoculation (see Figure 3 ).

[0040] Example 5: Detection of PBA-PU degradation by Pseudomonas sivasilis SCSIO 85030

[0041] Prepare PU solid medium with PBA-PU as the sole carbon source [mass fraction 1.5% PBA-PU, NaCl 2.2 g / L, Na2HPO4 2.8 g / L, (NH4)2SO4 0.5 g / L, CuCl2·2H2O 0.001 mg / L, H3BO3 0.03 mg / L, FeSO4.·7H2O0.2 mg / L, MnCl 2· 4H2O 0.003mg / L, NiCl2·6H2O 0.002mg / L, KH2PO4 1g / L, Na2EDTA0.5mg / L, CoCl2·6H2O 0.02mg / L, ZnSO4·7H2O 0.01mg / L, Na2MoO4·2H2O 0.003mg / L, agar 7.5g / L, solvent is water]. PBA-PU is synthesized from poly(1,4-butylene adipate PBA) with an average molecular weight of 2000 and 4'4'-methylene diphenylisocyanate (MDI). These two raw materials were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. The synthesized PBA-PU is a granular solid that can form turbid MSM culture medium at a concentration of 1g / L. Its chemical structure is shown in Figure 4 A. Inoculate Pseudomonas sylvaticus 85030 in PU solid medium with 1.5% PBA-PU as the sole carbon source, incubate inverted in the dark at 28°C for 6 days, and observe the production of hydrolysis circles. Figure 4 As shown in B, a transparent hydrolysis zone with a diameter of 24 mm was produced after 6 days of cultivation, which clearly showed the characteristics of producing extracellular hydrolases.

[0042] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement modes and are included in the scope of protection of the present invention.

[0043] SEQ ID NO.1

[0044]

Claims

1. Pseudomonas sivasensis SCSIO 85030, deposited with GDMCC No. 66488.

2. A microbial preparation, characterized in that The invention comprises the Pseudomonas assivasensis SCSIO 85030 according to claim 1 as an active ingredient.

3. Use of the Pseudomonas sivasilis SCSIO 85030 according to claim 1 or the preparation according to claim 2 in degradable plastics.

4. The use according to claim 3, characterized in that The plastic is water-soluble polyester polyurethane Impranil TM DLN and / or solid polyester polyurethane PBA-PU.

5. The use according to claim 3, characterized in that The application is that Pseudomonas sivasilis SCSIO 85030 produces extracellular degradation enzymes to degrade polyurethane plastics.

6. The use according to claim 5, characterized in that The degradative enzyme is protease or urease.

7. A method for degrading plastics, characterized in that: The method comprises using the Pseudomonas sivasilis SCSIO85030 described in claim 1 to degrade plastic.

8. The method according to claim 7, characterized in that The plastic is water-soluble polyester polyurethane Impranil TM DLN and solid polyester polyurethane PBA-PU.

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