CN116144508A - Cladosporium capable of degrading polyurethane plastics and application thereof - Google Patents
Cladosporium capable of degrading polyurethane plastics and application thereof Download PDFInfo
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- C12N1/14—Fungi; Culture media therefor
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
- B09B—DISPOSAL OF SOLID WASTE NOT OTHERWISE PROVIDED FOR
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- B09B3/60—Biochemical treatment, e.g. by using enzymes
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- C08J11/00—Recovery or working-up of waste materials
- C08J11/04—Recovery or working-up of waste materials of polymers
- C08J11/10—Recovery or working-up of waste materials of polymers by chemically breaking down the molecular chains of polymers or breaking of crosslinks, e.g. devulcanisation
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Abstract
The invention discloses a cladosporium cucumerinum strain capable of degrading polyurethane plastics and application thereof. A strain of cladosporium cucumerinum L-5 capable of degrading polyurethane plastics has a deposit number of GDMCCNo:62786. the cladosporium L-5 can grow by taking the Imprandill DLN as a unique carbon source, and has wide environmental adaptability. The degradation efficiency of the strain on polyurethane foam reaches 48% within 50 days, the volume of the foam plastic is obviously reduced after the strain is degraded, and the appearance is eroded. Therefore, the invention provides important biological resources for the biodegradation and utilization of the polyurethane waste plastics.
Description
Technical Field
The invention belongs to the field of applied microbiology, and relates to cladosporium and application thereof in degradation of polyurethane plastics.
Background
Plastic is a chemical material polymerized from synthetic or semi-synthetic compounds, has strong plasticity, and can be manufactured into various materials with different excellent physicochemical properties, so that the plastic is widely applied to the production and the life of human beings. From the invention of the plastic in the 50 th century of the 20 th century, about 90 hundred million tons of plastic are produced by human beings, and plastic products bring great convenience to human beings, meanwhile, the plastic abandoned in the environment is continuously accumulated, and great harm is caused to natural ecology and human society. Polyurethane (PU) is a polymer polymerized from a polyisocyanate and a polyol, wherein the polyisocyanate forms the hard segment of the polyurethane, imparting hardness and rigidity to the polyurethane material; the polyol forms the soft segment of the polyurethane and imparts elasticity to the polyurethane material. Adjusting the kinds and proportions of polyisocyanates and polyols for synthesizing polyurethane can produce polyurethane materials having various physicochemical properties, and thus polyurethane has been widely used in human society. Such as sofa in the field of household appliances, polyurethane foam for mattresses, building foam for construction engineering, plastic for laying plastic tracks, artificial skin in the medical field, etc.
While bringing convenience to polyurethane plastics, the unreasonable treatment also causes the problems of increasingly serious environmental pollution, resource waste and the like, and a reasonable treatment method is of great importance, wherein the depolymerization of plastics by utilizing a microorganism means is a greener and gentle effective way. The urethane bond contained in the long chain with high polymerization degree in the polyurethane is a key group for the biological depolymerization of polyurethane plastics. However, the high complexity and hydrophobicity of the true polyurethane synthetic plastic structure makes the waste polyurethane plastic difficult to degrade under natural conditions for a long period of time, directly resulting in difficulty in efficient screening of polyurethane degrading microorganisms. In recent years, with importance attached to plastic biodegradation at home and abroad, a plurality of PU degrading microorganisms have been reported, mainly including fungi such as Aspergillus flavus (202010879237.7), cladosporium sp.P7 (20201412031. X), saccharomyces (CN 202210403010.4) and bacteria such as Bacillus Brevibacillus sp.P10 (202011442856.6). At present, PU plastic biodegradation is still in a resource accumulation stage, and high-efficiency degradation microbial resources are fewer. Therefore, the PU plastic degrading microorganisms are further excavated, and the method has important research significance for realizing the biodegradation and resource utilization of waste plastics.
Disclosure of Invention
The invention provides Cladosporium sp.L-5 and application thereof in biodegradation of PU plastics.
Fungus L-5 of degradable polyurethane foam, classified and named as Cladosporium (Cladosporium) is deposited in the Guangdong province microorganism strain collection, the deposit address is Guangzhou, guangdong province institute of microorganisms, the deposit date is 2022, 9 months and 16 days, and the deposit number is GDMCCNo:62786.
the Cladosporium L-5 is prepared by taking a sample collected in a landfill as a material, adding an inorganic salt culture medium of 1% Imprandill DLN for enrichment culture, and obtaining a pure culture by utilizing dilution coating culture and observing a hydrolysis transparent ring. L-5 was identified as Cladosporium by strain ITS, actA sequence alignment analysis, the ITS, actA sequences were shown as SEQ ID NO.1 and SEQ ID NO. 2.
The invention relates to a culture, bacterial strain liquid, bacterial strain fermentation culture liquid or filtrate of fermentation culture liquid of Cladosporium L-5.
The cladosporium L-5 is a biological microbial agent with an active ingredient.
The invention relates to an application of Cladosporium L-5 and a culture, a bacterial strain bacterial solution, a bacterial strain fermentation culture solution or a filtrate of the fermentation culture solution thereof in biodegradation of polyester plastics, wherein the plastic substrates comprise ImpranlilDLN and real polyurethane plastics.
Wherein, the use concentration of the Impranlil DLN and the polyurethane foam plastic is respectively 2g/L and 5.7g/L, and the degradation conditions are as follows: inorganic salt culture medium, inoculating 10% spore with concentration of 1×10 6 Is used at 30 ℃ and 180rpm. Wherein the degradation efficiency of the Impranlin DLN for 26 hours is 22.89%, and the degradation efficiency of the polyurethane foam plastic 60d is 48.12%.
Advantageous effects
The invention adopts a solid culture medium containing 2 g/LIMPraniliDLN inorganic salt to successfully screen a strain L-5 with polyurethane degradation capability from the soil of a landfill, and the ITS sequence of the strain is aligned in NCBI, so that the result shows that the homology with Cladosporium (Cladosporium) is highest, and therefore, the Cladosporium is identified as Cladosporium. The strain adopted by the invention can degrade the water-based polyurethane Impranlil DLN in the growth process, and a transparent ring is generated on a 2g/LImpranil DLN inorganic salt solid culture dish. In addition, the strain has higher degradation effect on polyurethane foam. The Cladosporiumsp.L-5 has a great application potential in the aspect of biodegradation of waste polyurethane plastics based on a weight reduction experiment of polyurethane foam and the property characterization of polyurethane materials of the weight reduction experiment.
Drawings
FIG. 1 purification scheme of Strain L-5
FIG. 2 morphological observations of strain L-5 and phylogenetic tree of ITS, actA sequences
A: morphological characteristics of strain L-5; b: sequence phylogenetic tree based on ITSrDNA; c: phylogenetic tree based on actA sequence
FIG. 3 determination of the Imprandil DLN degrading Properties of the Strain
FIG. 4 measurement of degradation ability of polyurethane foam by Strain
a: weight loss of polyurethane foam by strain L-5 in 15 days and 50 days; b: SEM image of polyurethane foam; c: contact angle analysis of polyurethane foam
Biological material preservation information
Cladosporium (L-5) deposited at the microorganism strain collection center of Guangdong province at the accession number GDMCCNo, at the accession number of China, the institute of microbiology, academy of sciences, guangdong province, at the accession number of 2022, 9, 16 days: 62786.
Detailed Description
EXAMPLE 1 isolation and purification of strains
1.1 sample collection and preservation
Soil samples collected from the Anhui Xuancheng and other places are sealed in a sealed plastic package bag and stored at the temperature of minus 20 ℃.
1.2 enrichment of soil microflora
1.5g of the collected landfill environmental sample is weighed, the waste plastic sample is sheared by sterilized scissors, and 100mL of sterilized inorganic salt culture medium (0.7g.L) -1 K 2 HPO 4 ,0.7g·L -1 KH 2 PO 4 ，0.7g·L -1 MgSO 4 ·7H 2 O，0.005g·L -1 NaCl，1.34g·L -1 NH 4 Cl，0.002g·L -1 FeSO 4 ·7H 2 O，0.002g·L -1 ZnSO 4 ·7H 2 O，0.001g·L -1 MnSO 4 ·H 2 O), culturing at 28 ℃ and 180rpm for 20d; 5mL of the first enrichment was aspirated into 100mL of fresh inorganic salt medium containing 2 g/LIMPraniliDLN, 28℃and 180rpm for 20d.
1.3 separation and screening of PUR degrading bacteria by transparent circle method
The enriched liquid is processed according to 10 -1 、10 -2 、10 -3 、10 -4 、10 -5 、10 -6 Performing gradient dilution, coating on an inorganic salt solid culture dish containing 2 g/LIMPraniliDLN, culturing at 30deg.C for 3-4 days, and observing whether transparent rings are generated around colonies; single colonies with transparent circles were picked for streaking purification and Imprandil DLN degradation function verification was performed on inorganic salt solid dishes containing 2g/LImpranil DLN (FIG. 1).
Example 2 identification of strains
2.1 morphology of strains
The separated and screened strain L-5 grows in mycelium on an inorganic salt solid culture dish containing 2 g/LIMPraniliDLN, light green spores grow in the center of a colony, and transparent rings are arranged around colorless mycelia at the edge of the colony; PDA culture medium suitable for fungus culture(Potato 200 g.L) -1 ，20g·L -1 Glucose, 15 g.L -1 Agar), the L-5 colony grows in black green and fungus hypha shape; a large amount of mycelia and part of spores were produced during the growth of L-5 (FIG. 2A) as observed under an oil microscope.
2.2 identification
The ITSrDNA gene sequence of the strain is obtained by PCR amplification of the ITSrDNA of the strain, NCBI (www.ncbi.nlm.nih.govPblastP) is registered, and the ITS rDNA sequence of a typical strain similar to the experimental strain is obtained. The amplification primer is ITS1:5'-TCCGTAGGTGAACCTGCGG-3', ITS4:5'-TCCTCCGCTTATTGATATGC-3'. The sequencing results obtained were aligned with known sequences in the database using BLAST software in the GenBank database and analyzed by sequence alignment, found to be up to 100% identical to the Cladosporium meuconamia GUCC401.1 (OL 587465.1) (FIG. 2 b). The strain was thus initially identified as Cladosporiumsp, designated L-5. Then amplifying the strain actA sequence, wherein the amplification primer is ACT-512F: ATGTGCAAGGCCGGTTCGC, ACT-512R: TACGAGTCCTTCTGGCCCAT. The sequencing results obtained were aligned in the GenBank database using BLAST software with known sequences in the database and, by sequence alignment analysis, found to be up to 96% identical to the Cladosporium llicinumactA sequence (MH 102068.1) (FIG. 2C). Thus, this strain L-5 was identified as Cladosporium allium in combination with the above results. The strain L-5 and the strain P7 of the invention patent (202111412031. X) applied by Nanjing university of industry are cladosporium, and the strain P7 also has the degradation capability of polyurethane plastics. However, the highest ITS sequence identity was found by ITS sequence analysis to be 99.81% with the ITS sequence of Cladosporium cladosporidoides TJUJAN13 (OM 237126.1), so that strain P7 was initially identified as Cladosporium dendritic. Furthermore, the results of the evolution analysis showed (FIG. 2B) that the strain L-5 according to the invention was in a different evolutionary branch than the strain P7 according to the patent (202111412031. X). In addition, the optimal degradation temperature of the strain P7 on the Imprandil DLN is 30 ℃, and the optimal degradation temperature of the cladosporium L-5 on the Imprandil DLN is 28 ℃ (figure 3). The above results indicate that the Cladosporium p.L-5 according to the invention differs significantly from the strain P7 according to the invention patent (202111412031. X) and is not a fungus of the same kind.
EXAMPLE 3 degradation ability of the Strain to polyurethane foam
In order to study whether the strain L-5 can degrade the real PUR product, the commercial polyester PUR foam is selected as an experimental material, and the degradation performance of polyurethane plastics of the strain is determined through a plastic weight reduction experiment and the structural property change of the processed plastic. As shown in FIG. 4a, in the inorganic salt medium added with 0.1% yeast powder, 5.7g/L PUR foam was added, and the weight loss rates reached 25.65% and 48.32% after 15 days and 50 days, respectively. The treated plastic was selected for structural characterization for 50 days, and strain treatment was found to significantly disrupt the plastic surface structure, with the outer surface eroded, presumably due to hydrolysis by secreted degrading enzymes (fig. 4 b). Characterization of the hydrophobicity of the treated plastic revealed that strain treatment was able to significantly alter the hydrophilicity of the plastic with a greater contact angle (fig. 4 c). It was shown that strain L-5 may disrupt the intact structure of the foam by hydrolysis.
Claims (8)
1. Cladosporium sp.L-5, deposited at the microorganism strain collection center of Guangdong province, with a deposit address of Guangzhou, china, the university of Guangdong province, and a deposit date of 2022, 9 months and 16 days, with a deposit number of GDMCC No:62786.
2. the culture, bacterial strain solution, bacterial strain fermentation broth or filtrate of fermentation broth of Cladosporium L-5 as claimed in claim 1.
3. A biological agent comprising the cladosporium cucumerinum L-5 of claim 1 as an active ingredient.
4. A microbial inoculum according to claim 3 wherein the culture medium used to prepare the microbial inoculum is PDA culture medium or inorganic salt culture medium supplemented with 0.3% aqueous polyurethane.
5. Use of cladosporium L-5 as defined in claim 1 for biodegradation of polyurethane plastics.
6. The method according to claim 5, wherein the polyurethane has a concentration of 5-7g/L and a degradation condition of 28-35℃and 160-190rpm, and the strain is inoculated at 1% spore concentration of 0.5X10 7 -10×10 7 The degradation time is 45-50 days.
7. The method of claim 6, wherein the polyurethane has a concentration of 5.7g/L, the degradation condition is 30 ℃,180rpm, the strain inoculation amount is 1% and the spore concentration is 1X 10 7 The degradation time is 50 days.
8. Use of the microbial inoculum of claim 3 in the biodegradation of polyurethane plastics.
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| CN114644984A (en) * | 2022-04-28 | 2022-06-21 | 临沂大学 | Method for separating microorganism producing amylase from soil |
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| WO2020177175A1 (en) * | 2019-03-01 | 2020-09-10 | 中国科学院昆明植物研究所 | Fungal strain for degrading polyurethane plastic, culture method therefor and use thereof |
| CN112481137A (en) * | 2020-12-04 | 2021-03-12 | 南京工业大学 | Cladosporium and application thereof in degradation of polyurethane plastic |
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| Title |
|---|
| 岳晓禹;张华;陈威风;许文涛;郭明璋;: "应用高通量测序技术检测小麦储藏过程中真菌群落的变化", 食品科学, vol. 41, no. 14, pages 109 - 115 * |
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