Pseudomonas stutzeri SCSIO 85030 capable of degrading plastic and application thereof

Abstract

This invention discloses a strain of *Pseudomonas siliflorus* (…). Pseudomonas sivasensis SCSIO 85030 and its applications. *Pseudomonas sivatus* SCSIO 85030, with accession number GDMCC No. 66488, was deposited on June 9, 2025, at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), located at 5th Floor, Experimental Building, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou, Guangdong Province, 510070, China. This strain can produce various esterases, including extracellular urease and protease. When inoculated onto inorganic salt solid plates using waterborne polyurethane Impranil™ DLN and solid polyurethane PBA-PU as the sole carbon source, a clear hydrolysis zone appeared after 6 days. The *Pseudomonas sivatus* SCSIO 81030 of this invention has the ability to degrade polyurethane (PU) of varying structural complexity, providing theoretical guidance and technical support for the future recycling of polyurethane (PU).

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a highly efficient plastic-degrading Pseudomonas sivasensis strain SCSIO 85030 and its applications. Background Technology

[0002] Plastic is a synthetic polymer that has become an absolute necessity for human production and life since its inception, bringing revolutionary changes to human life, socio-economic development, and industries such as industry, agriculture, and medicine. However, while mass-produced plastics have brought great convenience to people's lives, they have also generated a large amount of waste. According to statistics, by 2015, approximately 8.3 billion tons of plastic had been generated globally, of which 6.3 billion tons had become waste. Of this, only 9% was recycled, 12% was incinerated, and 79% remained in landfills or the natural environment. Plastic waste is widely distributed, found in global terrestrial, marine, and even Antarctic ecosystems. Microplastic particles have been detected in animals, plants, and even the human body, posing a serious threat to ecosystem balance and human health. Polyurethane (PU) is a polymer synthesized by the reaction of diisocyanate, short-chain diols (carbon chain extenders), and polyols. Its structure contains repeating urethane bonds (-NHCOO-), and it is commonly used to make foam plastics, elastomers, fiber plastics, fibers, leather and shoe resins, coatings, adhesives, and sealants. Statistics show that polyurethane (PU) production accounts for 7.9% of global plastic production, ranking sixth in global plastic consumption. PU's structure contains amorphous soft segments and ordered hard segments. The hard segments are formed by the reaction of diisocyanates and short-chain diols, containing urethane bonds, while the soft segments are composed of polyester or polyether polyols, providing elasticity and flexibility. The strong polyurethane bonds, unique microstructure, and morphology of PU give this type of plastic extremely high durability, making it difficult to degrade naturally. Currently, the main methods of PU waste disposal are landfill and incineration, which are not only costly and unsustainable but also exacerbate carbon dioxide emissions and release large amounts 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 method of plastic treatment. Through the action of microorganisms, high-molecular-weight plastics are degraded into oligomers, which are ultimately mineralized into water and carbon dioxide. It offers advantages in terms of environmental friendliness and sustainability, effectively avoiding the severe environmental pollution problems caused by traditional waste disposal methods. Although strains from genera such as *Pseudomonas*, *Bacillus*, *Comamonas*, *Aspergillus*, *Cladosporium*, and *Penicillium* have been reported to degrade PU, the number of these strains is relatively small and their efficiency is generally low, severely limiting the application of biodegradation in PU plastic treatment. This patent addresses this by screening strains capable of simultaneously degrading waterborne polyester polyurethane—Impranil. TM The strains of DLN and solid polyester polyurethane—poly(1,4-butylene adipate)-based PU (PBA-PU)—were developed and applied to the degradation of PU waste in the natural environment, which has important application value for ecological and environmental protection. Summary of the Invention

[0004] The purpose of this invention is to address the problem of low degradation efficiency of existing PU biodegrading strains by providing a *Pseudomonas sivasensis* SCSIO 85030 strain with PU degradation capabilities. This bacterium can produce a variety of extracellular degrading 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 objective of this invention is to provide a strain of *Pseudomonas sivasensis* SCSIO 85030, with accession number GDMCC No. 66488. It was deposited on June 9, 2025, at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), located at: 5th Floor, Experimental Building, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou, Guangdong Province, 510070, China.

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

[0007] A third object of the present invention is to provide the use of the above-mentioned Pseudomonas sivasensis SCSIO 85030 or the above-mentioned formulation 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 involves Pseudomonas sivasensis SCSIO 85030 producing extracellular degradative enzymes to degrade polyurethane plastics.

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

[0011] The present invention also provides a method for degrading plastics, which uses *Pseudomonas siliflora* SCSIO 85030 to degrade plastics.

[0012] The *Pseudomonas sivasensis* SCSIO 85030 obtained in this invention can produce urease and protease. In the urease activity experiment, it produces a distinct red color on phenol red urea agar plates. In the protease activity experiment, it can liquefy gelatin in culture tubes.

[0013] Advantages of this invention:

[0014] This invention yielded *Pseudomonas sivasensis* SCSIO 85030, which produces extracellular urease and protease. This strain was inoculated with Impranil... TM On inorganic salt solid plates with DLN and PBA-PU as the sole carbon source, a clear hydrolysis zone appeared after day 6. These results indicate that *Pseudomonas sivasensis* SCSIO 85030 inhibits the growth of two polyurethanes (water-soluble polyurethane Impranil) with different structural complexities. TM Both DLN and solid polyurethane (PBA-PU) showed good degradation performance.

[0015] Pseudomonas sivasensis SCSIO 85030, accession number GDMCC No. 66488, was deposited on June 9, 2025, at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), located at: 5th Floor, Experimental Building, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou, Guangdong Province, 510070, China. Attached Figure Description

[0016] Figure 1 This image shows the colony morphology of Pseudomonas sivasensis SCSIO 85030 on LB solid medium.

[0017] Figure 2 Microscopic structure of Pseudomonas sivasensis SCSIO 85030.

[0018] Figure 3 and Figure 4 The bacteria were Pseudomonas sivasensis SCSIO 85030 and Impranil. TM Effect of PU degradation producing a transparent ring in an inorganic salt solid plate with DLN and PBA-PU as the sole carbon source (day 6 after inoculation).

[0019] Figure 5 This is an experimental plate for urease detection of Pseudomonas sivasensis SCSIO 85030.

[0020] Figure 6 The results of the gelatin liquefaction detection experiment of Pseudomonas sivasensis SCSIO 85030 are shown. From left to right, the first tube is the control group, and the third and fourth tubes are the experimental group. Detailed Implementation

[0021] The present invention will be further explained below with reference to the accompanying drawings and specific embodiments, but this does not constitute any limitation on the present invention. Unless otherwise specified, the reagents and methods used in the following embodiments are conventional in the art.

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

[0023] The strain 85030 described in this 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. 1g of sediment was weighed into a 250mL sterile shake flask in a clean bench, 100mL of sterile water was added, and after shaking well, 200μL of the suspension was taken and spread onto a substrate containing 1% Impranil. TM DLN was cultured in oligotrophic MSM inorganic salt solid medium at an incubator for one month. Single colonies producing clear zones were picked using an inoculation loop and transferred to LB medium supplemented with 5% (v / v) and 1% (v / v) Impranil. TMDLN was cultured on MSM inorganic salt solid medium, purified, and its species was determined. It was then preserved in 20% sterile glycerol tubes, and strain SCSIO85030 was obtained through screening.

[0024] MSM inorganic salt culture medium formula: 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, MnCl₂ 2· The following are the ingredients in the culture medium: 4H₂O 0.003 mg / L, NiCl₂·6H₂O 0.002 mg / L, KH₂PO₄ 1 g / L, Na₂EDTA 0.5 mg / L, CoCl₂·6H₂O 0.02 mg / L, ZnSO₄·7H₂O 0.01 mg / L, Na₂MoO₄·2H₂O 0.003 mg / L, with water as the solvent (pH 7.2). The solid culture medium is prepared by adding 15 g / L of agar. The preparation method involves mixing all components thoroughly, adjusting the pH, and sterilizing.

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

[0026] Example 2: Classification and identification of strain SCSIO 85030

[0027] (1) Colony morphology of strain 85030 screened in Example 1: On LB medium, the colonies of this strain were irregularly round. Initially, the colonies were white, turning pale yellow with increasing incubation time. 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: Strawberry strain 85030 was streaked onto LB agar plates and incubated at 28°C for 3 days. Purified single colonies were then picked and placed in 2 mL sterile EP tubes. DNA was extracted using the Chelx-100 method. Phylogenetic analysis was performed using the rDNA-ITS gene sequence. The rDNA-ITS gene PCR amplification primers were synthesized by Shanghai Sangon Biotech Co., Ltd. The 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 95℃ for 5 min; denaturation 94℃ for 30 s, annealing 56℃ for 30 s, extension 72℃ for 30 s, 35 cycles; final extension 72℃ for 5 min; storage at 4℃. PCR amplification products were detected by 1% agarose gel electrophoresis, and the PCR products were purified using an agarose gel DNA recovery kit (centrifuge column type). After purification, the PCR products were sent to Guangzhou Tianyi Huiyuan Co., Ltd. for sequencing, and the 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, this strain is defined as belonging to the kingdom Bacteria, phylum Pseudomonadota, class Gammaproteobacteria, order Pseudomonadales, family Pseudomonadaceae, genus Pseudomonas, and strain *Pseudomonas sivasensis* (SCSIO 85030). This bacterium is named *Pseudomonas sivasensis* SCSIO 85030, with accession number GDMCC No. 66488. It was deposited on June 9, 2025, at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), located at: 5th Floor, Experimental Building, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou, Guangdong Province, 510070, China.

[0033] Example 3: Enzyme activity experiment of Pseudomonas sivae SCSIO 85030

[0034] In the urease activity experiment, *Pseudomonas sibiricum* 85030 was inoculated onto a urease-phenol-red-urea solid medium plate and incubated at 28°C. The plate was observed every 24 hours to see if it changed 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 sivatus* 85030 was inoculated into gelatinase gelatin liquefaction solid slant medium, with three replicates (experimental groups). Gelatinase gelatin liquefaction solid slant medium (uninoculated) served as a blank control (control group). The culture was carried out at 28℃, and gelatin liquefaction was observed every 24 hours. After 7 days of inoculation, significant gelatin liquefaction was observed in the bacteria. Figure 6 ).

[0036] Formula for urease-phenol red-urea solid culture medium: peptone 1g / L, NaCl 5g / L, glucose 1g / L, KH2PO4 2g / L, phenol red 0.012g / L, agar 15g / L, 40% urea solution, solvent is water (pH 6.8). Preparation method: Mix all components thoroughly, adjust pH, and sterilize.

[0037] The formula for gelatinase-based gelatin liquefaction solid slant culture medium is as follows: peptone 5 g / L, glucose 20 g / L, gelatin 200 g / L, and water (pH 7.4) as the solvent. The preparation method involves mixing all components thoroughly, adjusting the pH, and sterilizing.

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

[0039] Preparation of waterborne polyurethane Impranil TM PU solid culture 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, MnCl2] 2· 4H₂O 0.003 mg / L, NiCl₂·6H₂O 0.002 mg / L, KH₂PO₄ 1 g / L, Na₂EDTA 0.5 mg / L, CoCl₂·6H₂O 0.02 mg / L, ZnSO₄·7H₂O 0.01 mg / L, Na₂MoO₄·2H₂O 0.003 mg / L, agar 7.5 g / L, solvent: water], wherein PU (Impranil TM DLN is a waterborne polyurethane, a commercially available anionic aliphatic colloidal polyester (PU) dispersion manufactured by Bayer AG, widely used in the textile and leather industries. *Pseudomonas sivatus* 85030 was inoculated into a PU solid medium with waterborne polyurethane as the sole carbon source and cultured upside down in the dark at 28°C for 6 days. The formation of hydrolysis zones was then observed. Figure 3 As shown, after 6 days of culture, a transparent hydrolysis zone with a diameter of 14 mm was produced, clearly demonstrating its characteristic of producing extracellular hydrolases. Test results showed that polyurethane in the culture medium was degraded, and the DLN solid culture base produced a clear transparent zone as early as day 6 after inoculation (see...). Figure 3 ).

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

[0041] Prepare a solid PU culture 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, FeSO4·7H2O 0.2 mg / L, MnCl2]. 2· The following ingredients were used: 4H₂O 0.003 mg / L, NiCl₂·6H₂O 0.002 mg / L, KH₂PO₄ 1 g / L, Na₂EDTA 0.5 mg / L, CoCl₂·6H₂O 0.02 mg / L, ZnSO₄·7H₂O 0.01 mg / L, Na₂MoO₄·2H₂O 0.003 mg / L, agar 7.5 g / L, and water as the solvent. PBA-PU was synthesized from poly(1,4-butylene adipate PBA) and 4',4'-methylene diphenylisocyanate (MDI) with an average molecular weight of 2000, purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. The synthesized PBA-PU was a granular solid that could form turbid MSM medium at a concentration of 1 g / L. Its chemical structure is shown in [link to chemical formula]. Figure 4 A. *Pseudomonas sivatus* 85030 was inoculated into PU solid medium with 1.5% PBA-PU as the sole carbon source and cultured upside down in the dark for 6 days. The formation of hydrolysis zones was then observed. Figure 4 As shown in Figure B, after 6 days of culture, a transparent hydrolysis zone with a diameter of 24 mm was produced, clearly demonstrating its characteristic of producing extracellular hydrolytic enzymes.

[0042] The above embodiments are preferred embodiments of the present invention, but the implementation of the present invention is not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

[0043] SEQ ID NO.1

[0044]

Claims

1. Pseudomonas stizasii (P. stizasii) Pseudomonas sivasensis ) SCSIO 85030, with the accession number GDMCC No. 66488.

2. A microbial preparation, characterized in that, Pseudomonas stutzeri comprising the polynucleotide of claim 1 Pseudomonas sivasensis ) SCSIO 85030.

3. The application of the *Pseudomonas sivatus* SCSIO 85030 of claim 1 or the formulation of claim 2 in degrading plastics, wherein the plastics are water-soluble polyester polyurethane Impranil™ DLN and / or solid polyester polyurethane PBA-PU.

4. The application according to claim 3, characterized in that, The application involves the production of extracellular degradative enzymes by *Pseudomonas sivatus* SCSIO 85030 to degrade polyurethane plastics.

5. A method for degrading plastics, characterized in that, The plastic is degraded by *Pseudomonas sivasioides* SCSIO85030 as described in claim 1, wherein the plastic is water-soluble polyester polyurethane Impranil™ DLN and solid polyester polyurethane PBA-PU.

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