Aeromicrobium ltx1 and application thereof in biodegradation of polyurethane plastic

Abstract

This invention discloses an aeromicrobacterium LTX1 and its application in the biodegradation of polyurethane plastics, belonging to the field of biodegradation technology. The degrading bacterium provided by this invention is named Aeromicrobacterium tamlense LTX1, and is deposited at the Guangdong Provincial Microbial Culture Collection Center, accession number GDMCC No: 62956. This strain was isolated and screened from coastal tidal flat soil in Cixi City, Ningbo City, Zhejiang Province. It can efficiently degrade polyurethane plastic foam, with a degradation efficiency of 90.37%, demonstrating significant application advantages and value.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to an aerobic microbacterium LTX1 and its application in the biodegradation of polyurethane plastics. Background Technology

[0002] Polyurethane (PUR), also known as polyurethane plastic, is a compound whose repeating structural unit, urethane, is formed by the condensation polymerization of isocyanate, polyol molecules, and chain extenders. Due to its superior flexibility, corrosion resistance, chemical resistance, elasticity, and ease of processing, polyurethane plastics are widely used in the construction, packaging, automotive, artificial leather, coating, and medical industries. Statistics show that in 2021, global plastic production reached 390 million tons, with polyurethane plastics accounting for 5.5% of the total production.

[0003] PUR plastics are mostly for single or short-term use, and currently account for 30% of all waste plastics. Traditional waste plastic treatment methods mainly include landfill, incineration, and recycling, but these methods have problems such as environmental pollution, high processing costs, and limited scope for secondary utilization. In contrast, bioremediation offers a new and more environmentally friendly approach to solving PUR plastic pollution.

[0004] Studies have found that microorganisms can survive and degrade polyurethane materials in soil contaminated with polyurethane. Microorganisms (especially bacteria and fungi) can rapidly degrade these synthetic polymers by secreting extracellular enzymes when under polyurethane pollution stress, thus achieving both material degradation and their own growth and metabolism. Although many bacteria and fungi capable of degrading PUR oligomers have been screened, reports on their degradation of commercially produced and used PUR materials are relatively few, and their degradation efficiency remains low. Therefore, screening for highly efficient PUR materials used in daily life is of great significance for protecting the environment and maintaining ecological balance. Summary of the Invention

[0005] Purpose of the Invention: Addressing the shortage of existing microbial resources for degrading PUR plastics, the purpose of this invention is to provide an actinomycete that efficiently degrades PUR plastics, and currently, no such genus has been reported to have the ability to degrade polyurethane foam. Another objective of this invention is its application in the harmless treatment, recycling, and environmental remediation of PUR plastic resources.

[0006] To achieve the above-mentioned objectives, the technical solution adopted by this invention is as follows:

[0007] A strain of Aeromicrobium tamlense LTX1 has been deposited at the Guangdong Provincial Center for Microbial Culture Collection on November 30, 2022, with accession number GDMCC No:62956.

[0008] The application of Aeromicrobium tamlense LTX1 in the degradation of polyurethane plastics.

[0009] The bacterial agent prepared from the aforementioned Aeromicrobium tamlense LTX1.

[0010] The application of the aforementioned microbial agent in the degradation of polyurethane plastics.

[0011] The method for preparing the bacterial agent is characterized in that the LTX1 strain is inoculated into an LB culture medium and cultured with shaking at 30°C for 24-26 hours to prepare a seed culture; the seed culture is then inoculated into an LB culture medium and cultured with shaking at 30°C until the logarithmic growth phase.

[0012] The method for degrading polyurethane plastics by Aeromicrobium tamlense LTX1 is characterized in that: the bacterial agent is applied to an inorganic salt culture medium containing polyurethane foam to be treated at an inoculation rate of 5-25%.

[0013] Beneficial effects: Compared with the prior art, the present invention screened a polyurethane degrading bacterium from the collected soil samples, which was identified as Aeromicrobium tamlense LTX1. This bacterium has the ability to degrade polyurethane plastics efficiently and can be used as a biodegrading agent in the harmless treatment, recycling and environmental remediation of PUR plastic resources. Attached Figure Description

[0014] Figure 1 The clear zone produced by strain LTX1 on Impranil DLN selection plates

[0015] Figure 2 The weight loss of PUR foam degraded by strains screened using the transparent zone method.

[0016] Figure 3 This is a colony morphology diagram of strain LTX1.

[0017] Figure 4 The results of Gram staining identification of LTX1 strain.

[0018] Figure 5 Phylogenetic tree of aerobic bacteria LTX1 based on 16S rDNA sequence

[0019] Figure 6 The weight loss of PUR foam degraded by LTX1 strain at different inoculum amounts

[0020] Figure 7 This is a scanning electron microscope image of LTX1 strain degrading PUR foam (A: CK; B: 5% inoculum; C: 15% inoculum).

[0021] Figure 8 This is the infrared spectrum of PUR foam degradation by strain LTX1.

[0022] Information on the preservation of biological materials

[0023] Aeromicrobium tamlense LTX1, classified as Aeromicrobium tamlense, 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 November 30, 2022, with accession number GDMCC No:62956. Detailed Implementation

[0024] The invention will now be described in detail with reference to specific examples and accompanying drawings.

[0025] Example 1: Screening of polyurethane-degrading strains

[0026] (1) Sample collection

[0027] Soil samples were collected from sandy soil of coastal tidal flats in Cixi City, Ningbo, Zhejiang Province, using a five-point sampling method. The samples were brought back to the laboratory and stored in a refrigerator at 4°C.

[0028] (2) Initial screening of degrading bacteria

[0029] Using the dilution coating plate method, 10 -3 10 -4 10 -5 Three concentration gradients of soil dilution were spread onto Impranil DLN screening plates and incubated upside down at 30°C for 6–7 days. Colonies with clear zones were selected, such as… Figure 1 The bacteria were purified by streak plating to obtain single colonies.

[0030] The composition (g / L) of the Impranil DLN screening plate is as follows: 0.92g K2HPO4·3H2O, 0.7g KH2PO4, 0.7g MgSO4·7H2O, 2.0g NH4Cl, 0.005g NaCl, 0.002g FeSO4·7H2O, 0.002g ZnSO4·7H2O, 0.001g MnSO4·H2O, 0.28% Impranil DLN, pH = approximately 7.2; 18-20g of agar powder is added to the solid culture medium.

[0031] (3) Secondary screening of degrading bacteria

[0032] Shake-flask culture: PUR foam was cut into 1×1×1.5cm pieces, dried in a 60℃ oven to constant weight (approximately 0.15g), and then added to the secondary screening medium for sterilization. Strains capable of degrading Impranil DLN were inoculated using an inoculation loop into 35mL / 50mL PUR foam secondary screening medium and cultured at 30℃ and 180rpm for 30 days. Three experimental groups and three uniform blank groups (aseptic inoculation) were also set up. After 30 days, the foam was removed and immersed in 5% (w / v) sodium hypochlorite solution for 4 hours. Mycelia attached to the foam were removed by ultrasonication, and the foam was then rinsed with ultrapure water and dried at 60℃ to constant weight. Mass loss was calculated. The strain named LTX1 showed the best degradation effect, with a weight loss of 68.90%. Figure 2 ).

[0033] The composition (g / L) of the above-mentioned secondary screening culture medium is as follows: 0.92g K2HPO4·3H2O, 0.7g KH2PO4, 0.7g MgSO4·7H2O, 2.0g NH4Cl, 0.005g NaCl, 0.002g FeSO4·7H2O, 0.002g ZnSO4·7H2O, 0.001g MnSO4·H2O, 1.0g Yeast Extract, pH = approximately 7.2.

[0034] Example 2

[0035] Morphological characteristics of aerobic microbe LTX1: LTX1 colonies on LB medium are yellow, round, raised, with neat edges, as shown in the image. Figure 3 As shown. LTX1 strain appears red upon Gram staining, indicating it is a Gram-negative bacterium, as shown in the results. Figure 4As shown. Phylogenetic analysis of the aerobic microbe LTX1: PCR amplification of the 16S rDNA of LTX1 strain yielded a fragment approximately 1.5 kb in length. After extraction and verification of the recombinant plasmid, sequencing revealed the full-length 16S rDNA of strain LTX1 to be 1542 bp. Highly homologous sequences were downloaded from the EzBioCloud database, and a phylogenetic tree of the strain was constructed using MEGA 7.0 based on Maximum Likelihood (ML) algorithm. Figure 5 It can be seen that LTX1 clusters on the same branch as Aeromicrobium tamlense SSW1-57 (DQ411541), with a sequence similarity of 99.79%. Based on morphological observation and physiological and biochemical characteristics, strain LTX1 is preliminarily identified as a strain of the genus Aeromicrobium sp. This strain was deposited at the Guangdong Provincial Microbial Culture Collection Center on November 30, 2022, with accession number GDMCC.No. 62956.

[0036] Example 3: Performance study of LTX1 strain in degrading PUR foam

[0037] PUR foam was cut into 1×1×1.5cm pieces, dried in a 60℃ oven to constant weight (approximately 0.15g), and then added to an inorganic salt culture medium for sterilization. LTX1 strain was inoculated into LB tubes and cultured to the logarithmic phase. 5%, 10%, 15%, 20%, and 25% bacterial suspension were then inoculated into 35mL / 50mL of inorganic salt culture medium containing PUR foam, respectively, and cultured at 30℃ and 180rpm for 10 days. Simultaneously, LB nutrients were added in the same proportion as the inoculum as a control treatment (aseptic inoculation). Each treatment was replicated in triplicate. After 10 days, the foam was removed and immersed in a 5% (w / v) sodium hypochlorite solution for 4 hours. Mycelia attached to the foam were removed by ultrasonication, and the foam was then rinsed with ultrapure water and dried at 60℃ to constant weight. The mass loss was calculated. The best degradation effect was observed when the inoculum was 15%, with a weight loss of 90.37%. Figure 6 ).

[0038] The composition (g / L) of the above inorganic salt culture medium is as follows: 0.92g K2HPO4·3H2O, 0.7g KH2PO4, 0.7g MgSO4·7H2O, 2.0g NH4Cl, 0.005g NaCl, 0.002g FeSO4·7H2O, 0.002g ZnSO4·7H2O, 0.001g MnSO4·H2O, pH = approximately 7.2.

[0039] Example 4: Characterization experiment of PUR foam surface using scanning electron microscopy (SEM)

[0040] SEM was used to observe changes in the surface and internal microstructure of PUR foam, thereby characterizing the degradation effect of strain LTX1 on PUR foam. When inoculated with 5% LTX1, the PUR foam skeleton structure became significantly thinner, looser, and fractured at multiple sites. When inoculated with 15%, the integrity of the PUR foam network structure was completely destroyed. In contrast, the control group's PUR foam skeleton structure remained regular and full. Figure 7 ).

[0041] Example 5: Fourier Transform Infrared Spectroscopy (FTIR) Analysis of Functional Group Changes in PUR Foam

[0042] FTIR analyzes the degradation ability of the LTX1 strain by measuring the absorption of light at different wavelengths based on the functional groups and chemical bond vibrations of PUR foam. Changes in functional groups and the appearance of new functional groups can prove that PUR foam has been degraded.

[0043] Compared to the control group, after 10 days of treatment with 5% and 15% LTX1, the carbonyl-C=O group (1725 cm⁻¹) showed improved carbonyl-C=O (1725 cm⁻¹) structure. -1 The characteristic peak corresponding to the ester bond was significantly weakened, and almost disappeared at an inoculum size of 10%, suggesting that the ester bonds in the material underwent some degree of breakage after treatment by this strain, and that extracellular esterase activity was present. The absorption peak of the COC stretching vibration of the ether bond in the degraded material was weakened (1174 cm⁻¹). -1 Compared to the control group and 5% inoculum, the absorption peak disappeared at 10% inoculum (the peak disappeared at this location), imino-NH- bending vibration (1531 cm⁻¹). -1 The weakening of the absorption peak indicates that the amide structure in the ether and urethane bonds of the material has undergone a certain degree of breakage. Figure 8 ).

Claims

1. A strain of Microbulbifer sp. LTX1, whose Latin name is Aeromicrobium tamlense , which is preserved in the Guangdong Microbial Culture Collection Center, with a preservation date of November 30, 2022, and a preservation number of GDMCC No: 62956.

2. The use of Aeromicrobium tamlense LTX1 in claim 1 in degrading polyurethane plastics.

3. A bacterial agent prepared from Aeromicrobium tamlense LTX1 in claim 1.

4. The use of the bacterial agent in claim 3 in degrading polyurethane plastics.

5. The method for preparing the bacterial agent according to claim 3, characterized by, The LTX1 strain is inoculated into LB culture medium and cultured at 30°C with shaking for 24-26 h to prepare a seed liquid; the seed liquid is inoculated into LB culture medium and cultured at 30°C with shaking until the logarithmic growth phase.

6. A method for degrading polyurethane plastic using the gas microbe LTX1 of claim 1, characterized by: The bacterial agent in claim 3 is added into inorganic salt culture medium containing polyurethane foam to be treated at an inoculation amount of 5-25%.

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