A strain capable of degrading PBAT plastic at room temperature and its applications

Abstract

This invention provides a strain of Aspergillus niger, F2, which can efficiently degrade PBAT under room temperature conditions. The strain is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences; accession number: CGMCC No. 40926; deposit date: November 17, 2023. Measurements showed that Aspergillus niger F2 achieved a 63% degradation rate of PBAT film after 56 days at 20–25°C; the degradation rate of PBAT powder reached 63% after 10 days and 91% after 30 days. This invention's Aspergillus niger F2 method for degrading PBAT plastics offers advantages such as high efficiency, simplicity, low cost, and environmental friendliness, demonstrating significant application potential in the degradation of PBAT agricultural films.

Description

Technical Field

[0001] This invention relates to the field of plastic degradation technology, and in particular to a strain that can efficiently degrade PBAT plastic at room temperature and its applications. Background Technology

[0002] While plastic products bring great convenience to human life, they also cause enormous environmental pollution due to their slow degradation. As a major producer and consumer of plastics, my country alone generated approximately 62 million tons of plastic waste in 2019. It is projected that by 2050, global cumulative plastic production will reach 34 billion tons, with approximately 300 million tons of plastic waste generated annually. The corresponding waste disposal will place immense pressure on global environmental governance.

[0003] To alleviate plastic pollution, my country has taken a number of measures, including actively promoting recyclable, easily recoverable, and biodegradable alternatives, and regulating the recycling of plastic waste. Among these, the use of biodegradable products, especially biodegradable plastic products, is an effective solution to address some of the pollution from single-use plastic products.

[0004] Currently, the most commonly used biodegradable plastics on the market include polybutylene terephthalate (PBAT), polylactic acid (PLA), and thermoplastic starch (TPS). PBAT is formed by the condensation polymerization of adipic acid, butanediol, and terephthalic acid. It contains long, flexible hydrocarbon chains and rigid side groups composed of aromatic benzene rings, exhibiting good ductility (elongation at break >700%). PBAT is a fully biodegradable aromatic polyester and is currently the most widely used biodegradable plastic, with its annual production increasing from 11,900 tons in 2011 to 33,800 tons in 2018. Based on comprehensive analysis of agricultural film development trends and plastic pollution control policies, the annual production of PBAT mulch film is expected to continue to grow and soon exceed 300,000 tons per year.

[0005] Although PBAT is a biodegradable material, its degradation effect is far from ideal in practical applications. The conjugated structure of the benzene ring in PBAT delocalizes the positive charge of the carbonyl carbon atom, reducing the sensitivity of the chemical bond to the hydrolysis process; at the same time, the steric hindrance effect of the aromatic structure reduces the accessibility of water molecules that can hydrolyze the chemical bond, thus making PBAT difficult to degrade and slow in degradation rate. If the PBAT mulch film laid during crop planting is not effectively degraded after use, the accumulation of waste PBAT mulch film in farmland will seriously affect the soil ecological environment and lead to crop yield reduction (Wu Qiang et al., 2017. Effects of PBAT biodegradable film covering on soil water and heat and yield of oasis drip-irrigated cotton. Transactions of the Chinese Society of Agricultural Engineering. 33(16), 135-143).

[0006] Currently, the main methods for treating biodegradable plastics are incineration, landfill, and industrial composting. Landfilling is simple to operate and can handle large volumes, but it requires a large amount of land and does not reduce the amount of waste or effectively treat the pollution source. Incineration significantly reduces the amount of waste, but it easily produces highly toxic dioxins, which pose a huge threat to the environment and human health. According to ISO 14855-1 / 2, industrial composting requires maintaining a composting temperature of 58℃ for a period of no more than 6 months. The amount of CO2 generated is used as the evaluation index for plastic degradation efficiency. Industrial aerobic composting can achieve good degradation effect of PBAT under a high temperature of (58±2)℃ (Ma Yichao et al., 2021. Degradation mechanism of PBAT-PLA biodegradable membrane bags in industrial aerobic composting in response to microorganisms. Transactions of the Chinese Society of Agricultural Engineering. 37(24), 224-231). However, maintaining high temperature requires a lot of energy and the reaction conditions are not easy to control. It is difficult to achieve the above-mentioned index conditions in general places.

[0007] Current research generally indicates that microbial degradation of PBAT at room temperature is ineffective. Huo Xiangdong et al. reported that after 60 days of cultivation at 30℃ and 150 rpm, the actual degradation rate of PBAT particles by PBAT-degrading bacteria reached 0.92% (Huo Xiangdong et al., 2017. Isolation, identification, and degradation capacity determination of polybutylene terephthalate (PBAT) degrading bacteria. Xinjiang Agricultural Sciences. 54(11), 2086-2091). Liu Jiaxi et al. isolated six PBAT-degrading strains, among which Pseudomonas sp. RD1-3 and Pseudomonas sp. N1-2 achieved degradation rates of 6.88% and 6.49% respectively after 8 weeks of degradation at 28℃ (Liu Jiaxi et al., College of Science, Northwest A&F University; College of Innovation and Experimentation, Northwest A&F University, 2021. Screening and degradation characteristics of PBAT mulch film degrading bacteria. Journal of Agricultural Environmental Science. 40(01), 129-136). Gao Zhiting et al. studied the effects of different soil conditions on the degradation characteristics of mulch films. The results showed that under the alternating day and night conditions of 20℃ (12h) and 40℃ (12h) for 90 days, the weight loss rates of the two PBAT mulch films were the highest, reaching 11.91% and 8.33%, respectively. However, at room temperature of 20℃, the weight loss rate of the two PBAT mulch films in soils with different moisture contents did not exceed 6.5% (Gao Zhiting et al., 2023. Response of mulch film degradation characteristics to different soil moisture and temperature conditions. Hubei Agricultural Sciences. 62(11), 27-33, 40). Based on current research, PBAT has a low degradation efficiency in soil at room temperature, and there is also a lack of microbial strains that can efficiently degrade PBAT. Summary of the Invention

[0008] In view of the above problems, the present invention provides a strain that can efficiently degrade PBAT in situ under normal temperature conditions.

[0009] The strain of Aspergillus niger described in this invention, which can efficiently degrade PBAT under room temperature conditions, is classified as Aspergillus niger and has the accession number F2. Aspergillus niger F2 is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences; accession number: CGMCC No. 40926; deposit date: November 17, 2023.

[0010] The Aspergillus niger F2 strain provided by this invention, which can efficiently degrade PBAT in situ under room temperature conditions, has colonies that are dark brown on the one side and light yellowish-brown on the other. The colony surface is flat with radial grooves, and the overall texture is distinctly velvety, with a small amount of colorless exudate. Figure 1 Mycelium and conidial heads can be observed under an optical microscope. Figure 2 ).

[0011] The present invention relates to the application of Aspergillus niger F2 in the degradation of PBAT plastic at room temperature.

[0012] The present invention relates to the application of Aspergillus niger F2 in the degradation of PBAT plastic at 20-25°C.

[0013] Furthermore, the aforementioned PBAT plastic is PBAT powder or PBAT film.

[0014] The Aspergillus niger F2 provided by this invention can efficiently degrade PBAT plastic at room temperature. Tests show that Aspergillus niger F2 degrades PBAT film for 28 days at 20-25°C with a degradation rate approaching 30%; the degradation rate of PBAT powder reaches 63% after 10 days. This invention's Aspergillus niger F2 degradation method for PBAT plastic has the advantages of high efficiency, simplicity, low cost, and environmental friendliness, and has great application potential in the degradation of PBAT agricultural film. Attached image description:

[0015] Figure 1 Colony morphology of Aspergillus niger F2 on solid culture medium.

[0016] Figure 2 Microscopic morphology of Aspergillus niger F2 under an optical microscope.

[0017] Figure 3 Image showing the results of ITS sequence alignment and identification of Aspergillus niger F2.

[0018] Figure 4 Comparison of the effects of Aspergillus niger F2 on the degradation of PBAT film at different times.

[0019] Figure 5Comparison of the appearance of PBAT film before and after degradation; where: A is the film before degradation, B is the film after 28 days of degradation, and C is the film fragments remaining after 56 days of degradation.

[0020] Figure 6 Comparison of the effects of Aspergillus niger F2 on the degradation of PBAT powder at different times.

[0021] Figure 7 Comparison of the appearance of PBAT powder before and after degradation; where A, B and C are the appearances before degradation, 10 days after degradation and 30 days after degradation, respectively.

[0022] Figure 8 XRD analysis of PBAT powder before and after degradation.

[0023] Figure 9 Comparison of PBAT powder before and after degradation using scanning electron microscopy; where A, C, and E are the powder surfaces before degradation, and B, D, and F are the powder surfaces 10 days after degradation.

[0024] The strain of Aspergillus niger described in this invention, which can efficiently degrade PBAT under room temperature conditions, is classified as Aspergillus niger and has the accession number F2. Aspergillus niger F2 is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences; accession number: CGMCC No. 40926; deposit date: November 17, 2023. Detailed Implementation

[0025] The technical solution of the present invention will be further described below with reference to various embodiments. Unless otherwise specified, the methods (materials) used in the present invention are all conventional technical means in the field.

[0026] Example 1

[0027] Strain screening: Soil samples were collected from the high-altitude region of Tibet. 3g of soil and PBAT powder were simultaneously added to 200mL of enrichment medium and cultured at 25℃ and 150r / min for 14 days with shaking. Then, 10mL of the culture was inoculated into 100mL of isolation medium and acclimated in a shaker at 25℃ for 30 days, with at least 5 passages. Finally, 1mL of the culture was used to prepare 10... -1 ~10 -6 Bacterial suspensions with concentration gradients were streaked onto PDA solid medium at a suitable gradient and incubated at 25°C for 3–7 days. Single colonies were then picked and inoculated onto PDA liquid medium for purification. After incubation, the culture was streaked onto PDA solid medium again. This purification process was repeated 3–5 times to obtain purified strains.

[0028] The components of the enrichment and isolation media used in the screening of the above strains are as follows:

[0029] Enrichment medium: PBAT powder 2.0 g / L, (NH4)2SO4 2.0 g / L, Na2HPO4 1.5 g / L, MgSO4 0.2 g / L, CaCl2 0.01 g / L, KH2PO4 1.5 g / L, pH natural.

[0030] Isolation medium: An inorganic salt basal medium with PBAT as the sole carbon source, prepared by adding 2g of PBAT powder or approximately 0.15g of PBAT film (10*10cm) to the following basic components: K₂HPO₄ 0.7g / L, KH₂PO₄ 0.7g / L, MgSO₄ . 7H2O 0.7g / L, (NH4)2SO4 0.005g / L, FeSO4.7H2O0.002g / L, ZnSO4.7H2O 0.002g / L, MnSO4.H2O 0.001g / L, pH 6.3~6.7.

[0031] ITS sequence identification of the strain: The purified strain obtained above was inoculated into PDA liquid medium and cultured at 25℃ and 150 r / min for 3-5 days with shaking. The bacterial cells were collected for genomic DNA extraction. Using the extracted genomic DNA as a template, PCR amplification of the ITS gene sequence was performed using the universal fungal identification primers ITS1 and ITS4. The reaction system was 20 μL: 0.5 μL upstream primer (10 μmol / L), 0.5 μL downstream primer (10 μmol / L), 10 μL Premix Taq™ (TaKaRa Taq™ Version 2.0 plus dye), 8 μL ddH2O, and 1 μL DNA template. PCR procedure: initial denaturation at 94℃ for 1 min, annealing at 57℃ for 2 min, extension at 72℃ for 1 min, for a total of 30 cycles, with a final extension at 72℃ for 10 min. After agarose gel electrophoresis, the amplified product was sent to a sequencing company for sequencing. The sequencing yielded an ITS gene sequence of 583 bp, with the GeneBank accession number OR234753.

[0032] Based on the sequencing results, a comparison was performed in a microbial species identification database, and the result showed the highest homology (92%) with the Aspergillus niger strain. Figure 3 This indicates that the strain obtained by screening in this invention is Aspergillus niger.

[0033] Example 2

[0034] Degradation effect of Aspergillus niger F2 on PBAT film: Prepare the same inorganic salt basal culture medium as in Example 1, with PBAT (film) as the sole carbon source.

[0035] The *Aspergillus niger* F2 strain obtained in Example 1 was fermented in PDA medium for 3 days and then inoculated into the aforementioned inorganic salt basal medium with PBAT film as the sole carbon source. A blank control was used with a medium without added bacterial solution. Each experiment was performed in triplicate. The culture medium was incubated at 25°C with shaking at 120 rpm. At 7, 14, and 28 days, the PBAT film was removed using sterile forceps, immersed in 100% ethanol for 30 minutes, washed at least three times with ultrapure water, and then dried at 65°C for 12 hours. The weight loss rate of the PBAT film was measured to assess the degradation rate, and the surface morphology differences before and after degradation were observed. The weight loss rate of the PBAT film was calculated using the following formula:

[0036] PBAT film weight loss (%) = (Initial mass of PBAT before treatment - Mass of PBAT after inoculation treatment) / Mass of PBAT before treatment × 100%

[0037] Test results are as follows Figure 4 As shown, at room temperature, the degradation rate of PBAT film by Aspergillus niger F2 reached 14.30%, 19.74%, 29.56%, and 63.44% at 7, 14, 28, and 56 days, respectively.

[0038] It should be noted that in addition to the biodegradable plastic components of Aspergillus niger F2, PBAT films also contain additives such as plasticizers, UV absorbers, antioxidants, and inorganic fillers (calcium carbonate, clay, etc.) to improve various properties and reduce costs. Most of these additives are recalcitrant and can affect the adhesion and degradation efficiency of the fungi on the film, leading to incomplete degradation and prolonging the degradation time. The morphology of the PBAT film before and after degradation is shown below. Figure 5 As shown: the film was intact and had a smooth surface before degradation. Figure 5 A), after 28 days of degradation, the film surface became rough and cracked, with more cracks and narrow fissures appearing. Figure 5 B). After further degradation for 56 days, the film was reduced to only discrete small fragments with relatively rough surfaces. Figure 5 C).

[0039] Example 3

[0040] Degradation effect of Aspergillus niger F2 on PBAT powder: Prepare the same inorganic salt basal culture medium as in Example 1, with PBAT (powder) as the sole carbon source.

[0041] The *Aspergillus niger* F2 strain obtained in Example 1 was fermented in PDA medium for 3 days and then inoculated into the above-mentioned inorganic salt basal medium with PBAT powder as the sole carbon source. A culture medium without added bacterial solution served as a blank control. Each experiment was repeated in triplicate. After culturing at 25°C and 120 rpm for 10 days, the PBAT powder was collected by filtration, washed at least three times with ultrapure water, dried at 65°C for 12 hours, and weighed. The weight loss rate of the PBAT powder was measured to assess the degradation rate. The surface and microstructure of the PBAT powder were also observed.

[0042] Degradation rate test results are as follows Figure 6 As shown, under normal temperature fermentation conditions, the degradation rate of PBAT powder by Aspergillus niger F2 reached 63.25% and 91.73% at 10 days and 30 days, respectively.

[0043] The morphology of PBAT powder before and after degradation is as follows: Figure 7 As shown: Before degradation, the powder showed no weight loss, a clean white surface, a smooth and compact texture, and no adhesion between powder particles. Figure 7 A); After 10 days of degradation, the powder weight decreased significantly, the surface turned pale yellow, and the texture became loose and rough. The powder particles were visibly adhered to each other. Figure 7 B). After 30 days of degradation, the powder is visibly sparse and loose, indicating that the powder has been almost completely degraded. Figure 7 C).

[0044] XRD analysis of PBAT powder before and after degradation: An appropriate amount of PBAT powder from Example 3 before and after degradation was naturally dried and then subjected to XRD analysis. The results are as follows... Figure 8 As shown, the characteristic peaks of PBAT are located at 16.1°, 17.26°, 20.72°, 23.14°, and 24.96°. The characteristic peaks at 16.1° and 17.26° show a significant forward shift, indicating a marked change in diffraction intensity after degradation, with varying degrees of change across different crystal planes. The diffraction peak intensities at 17.26° and 20.72° increase (degradation is faster in amorphous regions), while the diffraction peaks at 16.1°, 23.14°, and 24.96° decrease. Further calculation of crystallinity through integration reveals that the crystallinity before degradation was 37.38%, while the crystallinity after degradation was only 25.12%.

[0045] The scanning electron microscopy comparison results of PBAT powder before degradation and 10 days after degradation are as follows: Figure 9 As shown: The surface of PBAT plastic before degradation is relatively flat and smooth, with some tiny pores. Figure 9 A, C), after degradation, the surface becomes rough and locally uneven, with obvious grooves, depressions, pits and wrinkles, and large bulges ( Figure 9 B, D); Before degradation, the plastic surface often shows white spots (B, D); Figure 9After degradation (A, C), the white specks significantly decreased or disappeared. Figure 9 B, D). Observation at 100μm revealed that the undegraded plastic particles were larger and had a glossy appearance. Figure 9 E), after degradation, the powder particles become smaller, resulting in many fine powder particles, and the color becomes darker and duller. Figure 9 F).

Claims

1. A strain of Aspergillus niger that can degrade PBAT at room temperature ( Aspergillus niger F2, deposited at the China General Microbiological Culture Collection Center (CGMCC); accession number: CGMCC No. 40926; deposit date: November 17, 2023.

2. The application of Aspergillus niger F2 according to claim 1 in the degradation of PBAT plastic at room temperature.

3. The application of Aspergillus niger F2 according to claim 1 in the degradation of PBAT plastic at 20-25℃.

4. The application of Aspergillus niger F2 according to claim 1 in the degradation of PBAT plastic, characterized in that: The PBAT plastic is PBAT powder or PBAT film.

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