A polyurethane-based strain of Pseudomonas aeruginosa SCSIO 85019 that efficiently degrades it and its applications

Abstract

This invention discloses a strain of *Pseudomonas iranensis* SCSIO 85019 that efficiently degrades polyurethane plastics and its applications. *Pseudomonas iranensis* SCSIO 85019, with accession number GDMCC No. 66487, 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 esterases, ureases, and proteases. The strain was inoculated onto aqueous polyurethane Impranil... TM On inorganic salt solid plates with DLN and solid polyurethane PBA-PU as the sole carbon source, a clear hydrolysis zone appears after 6 days. The *Cladosporium oxysporum* SCSIO 81042 of this invention has the function of degrading 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 iranensis strain SCSIO 85019 and its applications. Background Technology

[0002] Polyurethane (PU) is a high-molecular-weight polymer synthesized by the reaction of diisocyanate, short-chain diols (carbon chain extenders), and polyols. Its structure contains repeating urethane bonds (-NHCOO-). PU is a segmented copolymer with rigid and flexible regions. The rigid portion is formed by the reaction of diisocyanate and short-chain diols, containing urethane bonds, while the flexible portion is composed of polyester-type or polyether-type polyols, providing elasticity and flexibility. By adjusting the ratio of diisocyanate and polyol, PUs with different thermal insulation properties, thermal conductivity, hardness, and corrosion resistance can be prepared. Therefore, PU can be widely used in the manufacture of various materials, such as soft foams (mattresses, sponges, interior decoration materials), rigid foams (sound insulation materials and other building materials), thermoplastics (sports shoes), and coatings (sealants, paints, adhesives). Statistics show that PU production accounts for 7.9% of global plastic production, ranking sixth in global plastic consumption. However, while large-scale PU production brings great convenience to people's lives, it also generates a large amount of waste. Because most polyurethane (PU) is a thermosetting polymer, its three-dimensional cross-linked structure makes it difficult to melt and dissolve. Therefore, only a small amount of PU waste is recycled through mechanical grinding, while the vast majority still relies on landfill and incineration. This traditional waste disposal method is not only costly and unsustainable, but also exacerbates carbon dioxide emissions and releases large amounts of microplastics and toxic substances into the environment, severely polluting the environment and threatening human health. For example, diamine compounds, degradation products of PU, are listed as "Substances of Very High Concern" by the European Chemicals Agency due to their carcinogenicity, mutagenicity, and reproductive toxicity. Furthermore, microplastics, once introduced into the marine environment, can adsorb organic pollutants and pathogens, and enter marine life and humans through the food chain, seriously threatening marine biodiversity and human health. Therefore, there is an urgent need to adopt green and sustainable methods to avoid the drawbacks of traditional PU plastic disposal methods.

[0003] Biodegradation is an environmentally friendly method of plastic disposal. Through the action of microorganisms, high-molecular-weight plastics are broken down 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. Currently, various bacterial and fungal strains have been reported to degrade polyurethane (PU), such as *Pseudomonas*, *Bacillus*, *Comamonas*, *Aspergillus*, *Cladosporium*, and *Penicillium*. However, these strains are primarily effective against impranilic acid. TM DLN exhibited good degradation activity. Impranil TM DLN is a water-based polyester-type polyurethane colloid existing in nanoparticle form with a particle size of 0.1 to 0.2 μM. It is a milky white liquid with a relatively simple structure; the hard segments are formed by the reaction of aliphatic diisocyanates and small-molecule chain extenders. In contrast, commercially available solid polyurethane plastics have a more complex structure (hard segments are typically formed by the reaction of aromatic diisocyanates and small-molecule chain extenders) and are insoluble in water. Therefore, Impranil... TM DLN is not representative of most commercially available solid polyurethane plastics. Furthermore, Impranil... TM The simple structure of DLN makes its carbamate bonds susceptible to hydrolysis by common proteases or lipases, resulting in generally low degradation efficiency of PU-degrading strains screened using this substrate for complex PU structures. Therefore, screening strains capable of degrading PUs of varying complexity is crucial for promoting the large-scale application of PU biodegradation. This patent identifies 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 iranensis* SCSIO 85019 strain with PU degradation capabilities. This strain can produce a variety of extracellular degrading enzymes, including esterases, ureases, and proteases, and can simultaneously 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 iranensis* SCSIO85019, with accession number GDMCC No. 66487. 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 this invention is to provide a microbial preparation comprising the aforementioned Pseudomonas iranensis SCSIO 85019 as an active ingredient.

[0007] A third objective of this invention is to provide the use of the aforementioned Pseudomonas iranensis SCSIO85019 or the aforementioned formulation in degrading plastics.

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

[0009] Specifically, the application involves Pseudomonas iranensis SCSIO 85019 producing extracellular degradative enzymes to degrade polyurethane plastics.

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

[0011] The present invention also provides a method for degrading plastics using Pseudomonas iridis SCSIO 85019.

[0012] Preferably, the plastic is water-soluble polyester-type polyurethane Impranil. TM DLN and / or solid polyester polyurethane PBA-PU.

[0013] The *Pseudomonas iranensis* SCSIO 85019 obtained in this invention can produce esterases, proteases, and ureases. In esterase activity experiments, it produces opaque white halos on Tween 20 and Tween 80 plates. In protease activity experiments, it can liquefy gelatin in culture tubes. In urease activity experiments, it produces a distinct red color on phenol red urea agar plates.

[0014] Advantages of this invention:

[0015] This invention yielded *Pseudomonas iranensis* SCSIO 85019, which produces extracellular esterases, ureases, and proteases. 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 aeruginosa* 85019 inhibits the growth of two polyurethanes (water-soluble polyurethane Impranil) with different structural complexities. TN Both DLN and solid polyurethane (PBA-PU) showed good degradation performance.

[0016] Pseudomonas iranensis SCSIO 85019, with accession number GDMCC No. 66487, 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

[0017] Figure 1 This image shows the colony morphology of Pseudomonas iranensis SCSIO 85019 on LB solid medium.

[0018] Figure 2 Microscopic structure of Pseudomonas iranensis SCSIO 85019.

[0019] Figure 3 and Figure 4 These are Pseudomonas iranensis SCSIO 85019 and Impranil. TM Effect of DLN, PBA-PU, and PU as the sole carbon source in an inorganic salt solid plate to produce a transparent ring due to PU degradation (day 6 after inoculation).

[0020] Figure 5 This is an experimental plate for the detection of urease in Pseudomonas iranensis SCSIO 85019.

[0021] Figure 6 Figure 1 shows experimental plates for the esterase detection of Pseudomonas iranensis SCSIO 85019. Figure 2 shows an experimental plate with Tween 20 as the substrate and Figure 3 shows an experimental plate with Tween 80 as the substrate.

[0022] Figure 7 The results of the gelatin liquefaction detection experiment for Pseudomonas iranensis SCSIO 85019 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

[0023] 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.

[0024] Example 1: Isolation and preservation of strain SCSIO 85019

[0025] The 85019 sample described in this invention was isolated from marine sediments in Daya Bay, Huizhou. The sample was collected on October 10, 2024, at 22°43.43'N, 114°42.76'E. 1 g of sediment was weighed into a 250 mL sterile shake flask in a clean bench, 100 mL of sterile water was added, and the mixture was shaken well. 200 μL of the suspension was then 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 a clear zone were picked using an inoculation loop and transferred to LB medium supplemented with 5% (v / v) and 1% (v / v) Impranil. TM DLN was subcultured on MSM solid medium, purified, and its species determined. It was then cultured in 20% sterile glycerol tubes for preservation, and strain 85019 was obtained through screening. The MSM inorganic salt medium formula was: 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.

[0026] 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.

[0027] Example 2: Classification and identification of strain SCSIO 85019

[0028] (1) Colony morphology of strain 85019 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 ).

[0029] (2) Molecular biological identification

[0030] DNA extraction: Strawberry strain 85019 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'-TACGGCTACC TTGTTACGACTT-3'. The PCR reaction system was as follows:

[0031] 2×GS Taq PCR Mix 25μL Primer 1 (10μM) 2.0μL Primer 2 (10μM) 2.0μL Template DNA 2.0μL <![CDATA[ddH2O]]> 19μL Total volume 50μL

[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 iranensis*, 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 iranensis (Pseudomo nas iranensis SCSIO 85019), and is named Pseudomonasiranensis SCSIO 85019.

[0033] Pseudomonas iranensis SCSIO 85019, with accession number GDMCC No. 66487, 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.

[0034] Example 3: Enzyme activity experiment of Pseudomonas iridis SCSIO85019

[0035] In the urease activity experiment, *Pseudomonas iridis* SCSIO 85019 was inoculated onto urease-phenol-red-urea solid medium plates and incubated at 28°C. The plates were observed every 24 hours to see if they changed from yellow to red. On the 4th day after inoculation, the center of the plate turned red. Figure 5 ).

[0036] In the esterase activity experiment, *Pseudomonas iridozoae* SCSIO 85019 was inoculated onto esterase agar plates containing Tween 20 and Tween 80, and incubated at 28°C. The presence of a white opaque halo was observed every 24 hours. On the 4th day after inoculation, white opaque halos were observed on both Tween 20 and Tween 80 esterase agar plates. Figure 6 ).

[0037] In the protease experiment, *Pseudomonas iridis* SCSIO 85019 was inoculated into gelatinase gelatin liquefaction slant medium, with three replicates (experimental groups). Gelatinase gelatin liquefaction slant medium (without inoculation) served as a blank control (control group). The culture was carried out at 28°C, and gelatin liquefaction was observed every 24 hours. On day 7 after inoculation, significant gelatin liquefaction was observed in the bacteria. Figure 7 ).

[0038] The formula for esterase agar solid medium is as follows: peptone 1 g / L, NaCl 5 g / L, CaCl2 0.1 g / L, agar 9 g / L, Tween (Tween 20 and Tween 80) 10 mL / L, and water (pH 7.4) as the solvent. The preparation method involves mixing all components thoroughly, adjusting the pH, and sterilizing.

[0039] 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.

[0040] 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.

[0041] Example 4: Degradation of Impranil by Pseudomonas aeruginosa SCSIO 85019 TM DLN detection

[0042] 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 aeruginosa* SCSIO 85019 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 16 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 ).

[0043] Example 5: Detection of PBA-PU degradation by Pseudomonas iridis SCSIO 85019

[0044] A solid PU culture medium with PBA-PU as the sole carbon source was prepared [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 iridis* SCSIO 85019 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 20 mm was produced, clearly demonstrating its characteristic of producing extracellular hydrolytic enzymes.

[0045] 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.

[0046] SEQ ID NO.1

[0047]

Claims

1. *Pseudomonas aeruginosa* ( Pseudomonas iranensis ) SCSIO 85019, accession number GDMCCNo.66487.

2. A microbial preparation, characterized in that, Contains the *Pseudomonas aeruginosa* as described in claim 1 ( Pseudomonasiranensis SCSIO 85019 is used as the active ingredient.

3. The *Pseudomonas aeruginosa* as described in claim 1 (… Pseudomonas iranensis The application of the microbial preparation of claim 2 or SCSIO 85019 in the degradation of plastics, wherein the plastic is a water-soluble polyester polyurethane Impranil™ DLN and / or a solid polyester polyurethane PBA-PU.

4. The application according to claim 3, characterized in that, The application is for *Pseudomonas aeruginosa* ( Pseudomonas iranensis SCSIO 85019 produces extracellular degradative enzymes to degrade polyurethane plastics.

5. The application according to claim 4, characterized in that, The degrading enzyme is an esterase, protease, or urease.

6. A method for degrading plastics, characterized in that, The plastic is degraded by Pseudomonas iridis SCSIO85019 as described in claim 1, wherein the plastic is water-soluble polyester polyurethane Impranil™ DLN and / or solid polyester polyurethane PBA-PU.

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