Scopulariopsis brevicaulis and application thereof

Abstract

This invention discloses a strain of *Pleurotus erythrorhizon* and its application in the degradation of polyurethane plastics. A strain of *Pleurotus erythrorhizon*, SC23, capable of degrading polyurethane foam, has the accession number GDMCC No: 63578. The *Pleurotus erythrorhizon* SC23 described in this invention can grow using Impranil DLN as the sole carbon source, exhibiting broad environmental adaptability and plastic degradation potential. This strain achieves a degradation efficiency of 94.17% for 6 g / L polyurethane foam within 20 days and a degradation efficiency of 37.86% for 1 g / L agricultural mulch film (PBAT) within 30 days. After degradation by the strain, the volume of the plastic is significantly reduced, and the plastic surface is eroded. Therefore, this invention provides an important biological resource for the biological treatment of waste polyurethane plastics and agricultural mulch film.

Description

Technical Field

[0001] This invention belongs to the field of applied microbiology and relates to a strain of Cladosporium and its application in degrading polyurethane plastics and agricultural mulch film PBAT. Background Technology

[0002] Plastics are products made primarily of organic polymers, with plasticizers, stabilizers, or additives as auxiliary elements, and molded using basic chemical methods. In the early 20th century, the uses of plastics were widely explored, and they have now become a major global commodity, experiencing exponential growth in modern society and permeating almost every aspect of human life. Polyurethane (PU) is a polymer composed of polyisocyanates and polyols. Polyisocyanates form the hard segments of polyurethane, giving the material its hardness and rigidity; polyols form the soft segments, giving the material its elasticity. Polyurethane (PU) has wide applications in household, industrial, and medical fields. Its unique structure gives it high tensile strength, high hydrolysis resistance, and a high melting point. It is used in adhesives, foams, food-grade coatings, insulation, tires, sponges, and many other products. Statistics show that global polyurethane production in 2016 was approximately 22.3 million tons, with an annual growth rate of 4.0%. In Europe alone, polyurethane plastics accounted for nearly 7.7% of total plastic demand. In Australia, approximately 2.5 million tons of plastic waste were generated in 2016-2017, of which only 12% was recycled, and 87% was dumped in landfills. Discarding plastic waste causes serious ecological pollution and is also a waste of carbon resources.

[0003] Currently, the main methods for recycling waste plastics include sorting and recycling, physical and chemical treatment for degradation, and incineration for power generation. In recent years, utilizing microorganisms to achieve the biodegradation of plastics and the resource-based upgrading and utilization of degradation products has become an optional means of plastic resource utilization. Plastic biodegradation and resource utilization align with the requirements of national green development. In recent years, with increasing attention paid to plastic biodegradation both domestically and internationally, several polyurethane plastic degrading microorganisms have been reported, mainly including fungi such as *A. flavus* G10 (CN109762744A), *Aspergillus flavus* (202010879237.7), *Cladosporium* sp. P7 (202011412031.X), and yeasts such as *CN202210403010.4*, as well as bacteria such as *Brevibacillus* sp. P10 (202011442856.6). However, limited by low degradation efficiency, scarce biodegradable microorganisms, and a lack of advanced degradation processes, the biodegradation of polyurethane plastics is currently still in the resource accumulation and laboratory research stage, with a limited number of highly efficient degrading microorganisms. Therefore, further exploration of highly efficient polyurethane-degrading microorganisms using a combination of evaluation methods is of great significance for realizing the biodegradation and resource utilization of polyurethane plastics. Summary of the Invention

[0004] This invention provides a strain of Sarocladium sp. SC23 and its application in the biodegradation of polyurethane plastics.

[0005] A fungus SC23, which can degrade polyurethane foam, is classified and named *Sarocladium sp.*. It is deposited at the Guangdong Provincial Culture Collection Center for Microbial Cultures, located at the Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, on June 21, 2023. Its accession number is GDMCC No: 63578.

[0006] The *Cladosporium* SC23 strain described in this invention was cultured using soil collected from coniferous forests in Yichun, Yunnan Province, in an inorganic salt medium supplemented with 2 g / L limpurnil DLN. The presence of polyurethane degradation function was determined by hydrolysis of the clear zone. Pure cultures of the strain were then isolated and purified. Preliminary identification of SC233 as *Cladosporium* was achieved through 18S rRNA sequence alignment analysis, as shown in SEQ ID NO. 1.

[0007] The present invention relates to the culture, bacterial culture, fermentation broth, or filtrate of the fermentation broth of *Cladosporium* SC23.

[0008] The *Cladosporium* SC23 described in this invention is a biological agent with active ingredients.

[0009] The application of the *Cladosporium* SC23 and its culture, bacterial culture, fermentation broth, or filtrate of fermentation broth in the biodegradation of plastics, as described in this invention.

[0010] As a preferred embodiment of the present invention, the plastic is selected from Impranil DLN, PBA-PU, real polyurethane PUR plastic, and agricultural mulch film PBAT, etc.

[0011] As a further preferred embodiment of the present invention, the degradation conditions are an inorganic salt culture medium, and the inoculation is carried out at a spore concentration of 2% and 1×10⁻⁶. 6 Under conditions of spore suspension at 30℃ and 180 rpm, the weight loss rate of polyurethane (PUR) foam with a concentration of 6 g / L was 94.17% within 20 days; the weight loss rate of agricultural mulch film (PBAT) with a concentration of 1 g / L within 30 days was 37.86%.

[0012] Beneficial effects

[0013] This invention successfully screened a polyurethane-degrading strain, SC23, from coniferous forest soil in Northeast China using a solid culture medium containing 2 g / L Impranil DLN inorganic salts. The 18S rRNA sequence of the strain was compared with that of *Sarocladium* sp. in NCBI, showing the highest homology, and was therefore identified as *Sarocladium* sp. SC23. During its growth, the strain used in this invention was able to degrade aqueous polyurethane Impranil DLN and PBA-PU, producing a transparent zone on solid culture dishes containing 2 g / L Impranil DLN or 1 g / L PBA-PU inorganic salts. Furthermore, this strain exhibited high degradation efficiency for polyurethane (PUR) foam and agricultural mulch film (PBAT). Based on weight loss experiments and characterization of the properties of the degraded plastics in polyurethane (PUR) foam and agricultural mulch film (PBAT), it is evident that *Sarocladium* sp. SC23 involved in this invention has significant application potential in the biodegradation of waste plastics. Attached Figure Description

[0014] Figure 1 Purification process of strain SC23

[0015] Figure 2 Morphological observation and phylogenetic tree of 18S rRNA sequence of strain SC23

[0016] a: Morphological characteristics of strain SC23; left image shows colony morphology; right image shows cell observation under oil immersion (100×). b: Phylogenetic tree analysis of the 18S rRNA sequence of strain SC23.

[0017] Figure 3 Determination of the degradation properties of Impranil DLN and PBA-PU by the strain

[0018] Figure 4 Determination of the strain's ability to degrade polyurethane foam

[0019] a: Weight loss rate of polyurethane foam by strain SC23 over 20 days; b: SEM image of polyurethane foam.

[0020] Figure 5 Determination of the strain's ability to degrade PBAT agricultural film

[0021] Information on the preservation of biological materials

[0022] Sarocladium sp.SC23, classified as Sarocladium sp., is deposited at the Guangdong Provincial Microbial Culture Collection Center, located at the Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, on June 21, 2023. Its accession number is GDMCC No: 63578. Detailed Implementation

[0023] Example 1. Isolation and purification of polyurethane plastic degrading strains

[0024] Soil samples collected from coniferous forests in Yichun were sealed in airtight plastic bags and stored at -20℃. 0.25g of the collected Northeast coniferous forest soil sample was weighed and placed in 100mL of inorganic salt medium (0.7g·L⁻¹) containing 2g / L Impranil DLN. - 1 K2HPO4, 0.7 g·L -1 KH2PO4, 0.7 g·L -1 MgSO4·7H2O, 0.005 g·L -1 NaCl, 1.34 g·L -1 NH4Cl, 0.002 g·L -1 FeSO4·7H2O, 0.002 g·L -1 ZnSO4·7H2O, 0.001 g·L -1 In MnSO4·H2O, cultured at 30℃ and 180 rpm for 7 days. The enrichment solution was then processed according to a 10... -1 10 -2 10 -3 10 -4 10 -5 10 -6 10 -7 10 -8Perform serial dilutions and spread onto LB medium supplemented with 5% (v / v) yeast extract (5 g / L). -1 10g·L -1 Tryptone, 10 g / L -1 In an inorganic salt culture medium plate containing NaCl and 2 g / L Impranil DLN, incubate at 30°C for 3-4 days, observing whether a clear zone forms around the colony. Single colonies with clear zones are picked and streaked for purification, and their Impranil DLN degradation function is verified on an inorganic salt solid culture dish containing 2 g / L Impranil DLN. Figure 1 The results showed that one of the filamentous fungi produced the largest clear zone by degrading DLN, and it was named SC23.

[0025] Example 2. Morphological observation and identification of polyurethane plastic degrading strain SC23

[0026] The isolated strain SC23 exhibited hyphal growth on inorganic salt solid culture dishes containing 2 g / L Impranil DLN, with white spores at the center of the colonies and a clear halo around the white hyphae at the colony edge; it also showed growth on PDA medium (200 g / L) suitable for fungal culture. -1 Potatoes, 20g·L -1 Glucose, 15 g·L -1 On agar, SC23 colonies appear white with fungal mycelial growth; under an oil immersion microscope, it was observed that SC23 produced a large amount of mycelium and some spores during its growth. Figure 2 a).

[0027] The 18S rRNA gene sequence of this strain was obtained by acquiring a draft genome map. The sequencing results were then compared with known sequences in the GenBank database using BLAST software via NCBI (www.ncbi.nlm.nih.gov / PblastP). Sequence alignment analysis revealed that it is most closely related to *Sarocladium* sp. Figure 2 b). Therefore, the strain was preliminarily identified as Sarocladium sp.

[0028] Example 3. Degradation ability of the strain against Impranil DLN and PBA-PU

[0029] To investigate whether strain SC23 involved in this invention has the ability to degrade Impranil DLN and PBA-PU, strain SC23 (1×10⁻⁶) was inoculated into oligotrophic plates (containing 5% LB inorganic salt medium) supplemented with 2 g / L Impranil DLN or 1 g / L PBA-PU. 6A spore suspension (5 μL) was incubated at 25°C, and the presence of a clear zone was observed periodically. After 3 days, a clearly visible clear zone appeared around the colony of strain SC23, indicating that strain SC23 could degrade the substrate Impranil DLN. Figure 3 a) and PBA-PU( Figure 3 b).

[0030] Example 4. The ability of the strain to degrade polyurethane foam

[0031] To investigate whether the strain SC23 involved in this invention has the ability to degrade actual polyurethane plastic products, commercially available polyester-type PUR foam was selected as the experimental material. The polyurethane plastic degradation performance of strain SC23 was determined through plastic weight reduction experiments and changes in the structural properties of the plastic itself after strain treatment. In an inorganic salt medium containing 5% (v / v) LB, 6 g / L of PUR foam was added, and 2% spores were inoculated at a concentration of 1×10⁻⁶. 6 The spore suspension, cultured at 30℃ and 180 rpm, showed weight loss rates of 30.41% and 94.17% after 5 and 20 days, respectively. Figure 4 a). Structural characterization was performed on plastics treated for 20 days, revealing that the bacterial strain treatment significantly damaged the plastic surface structure, with the outer surface being eroded. Figure 4 b) It is speculated that strain SC23 can produce plastic-degrading enzymes that destroy surface structures.

[0032] Example 5. Degradation ability of the strain on PBAT agricultural mulch film

[0033] To investigate whether the strain SC23 involved in this invention can degrade actual agricultural mulch film PBAT, commercially available PBAT agricultural mulch film was selected as the experimental material. The PBAT degradation performance of the strain was determined through a plastic weight reduction experiment. Figure 5 As shown, in an inorganic salt medium containing 5% (v / v) LB, 1 g / L of PBAT film was added, and 2% of strain SC23 spores were inoculated at a concentration of 1×10⁻⁶. 6 The spore suspension was cultured at 30°C and 180 rpm for 30 days. After that, the PBAT film showed fragmentation. Figure 5 a) The weight loss rate reached 37.86% ( Figure 5 b). This indicates that strain SC23 has good degradation ability on PBAT films.

Claims

1. A broom mold that can degrade polyurethane foam Sarocladium sp. SC23 is deposited at the Guangdong Provincial Center for Microbial Culture Collection, located at the Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, on June 21, 2023. Its accession number is GDMCC No: 63578.

2. The *Cladosporium* SC23 of claim 1 is a biological agent containing active ingredients.

3. The application of the *Cladosporium SC23* according to claim 1 or the biological agent according to claim 2 in the degradation of plastics, wherein the plastic is selected from polyurethane foam (PUR) or agricultural mulch film (PBAT).

4. The application according to claim 3, characterized in that, The polyurethane foam PUR concentration was 6 g / L, the degradation conditions were 30 °C, 180 rpm, the Scytalidium candidum SC23 strain inoculation amount was 2% spore concentration was 1 x 10 6 of the seed liquid, and the degradation time was 20 days.

5. The application according to claim 3, characterized in that, The PBAT concentration was 1 g / L, the degradation conditions were 30 ℃, 180 rpm, the inoculum size of *Cladosporium* SC23 strain was 2%, and the spore concentration was 1×10⁻⁶. 6 The seed liquid has a degradation time of 30 days.

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