Pristerhina arctica jou-s14 and use thereof

Abstract

The application discloses a pristinamycin I Jou-S14 and application thereof, the strain has been preserved in the China General Microbiological Culture Collection Center on March 14, 2025, and the preservation number is CGMCC No.33815, the weight loss rate of the strain to hcPET-MPs and high crystallinity PET film is 5.02+0.21% and 7.09+0.35% respectively within 30 days, and the strain shows 1-2% weight loss effect to PA-MPs, PP-MPs, HDPE-MPs and PS-MPs and various microplastics.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of environmental microbiology, and particularly relates to a Pristina aritica strain Jou-S14 and application thereof. BACKGROUND

[0002] With the continuous increase of global plastic production, polyethylene terephthalate (PET) as a commonly used thermoplastic polyester material is widely used in packaging, bottled water, textile fibers and food industry. A large amount of PET waste enters the natural environment, and after long-term accumulation and gradual fragmentation into PET microplastics (PET-MPs), especially in the marine environment, due to its non-degradability and high crystallinity structure, it can maintain chemical stability on a multi-year scale, thereby causing serious ecological and environmental risks. A large number of studies have shown that the high crystalline structure of PET makes it difficult for enzymes or microorganisms to attack the internal ester bond, which is the main factor limiting the efficiency of microbial degradation. Therefore, the resources of microorganisms that can directly use PET waste or PET microplastics in the environment as the sole carbon source for growth are very scarce, and the screening of related strains usually needs to go through a complex and long-term enrichment and strict screening process.

[0003] Current studies have reported microorganisms and enzymes that can degrade low-crystallinity PET films or artificially modified PET materials, such as PETase from Ideonella sakaiensis. However, these strains are usually difficult to degrade high-crystallinity (> 30%) PET waste that actually exists in the natural environment, and their degradation ability often depends on the pretreatment of PET, such as heat treatment, surface sanding or solvent swelling, etc. Therefore, it is of great scientific and application significance to screen natural strains that can adapt to complex conditions such as high salt, temperature fluctuation, ultraviolet radiation and coexistence of various plastics, and have the potential to degrade high-crystallinity PET, for environmental microplastic pollution control and discovery of new ester hydrolases.

[0004] Coastal sediments and seawater-plastic interface biofilms are important reservoirs of plastic-degrading microorganisms in nature. PET waste exposed to the marine environment for a long time often forms a complex microbial colonization system on its surface, which contains a large number of potential functional bacterial populations with oxidoreductase, ester hydrolase and aromatic compound metabolism capabilities. Therefore, by directional enrichment of marine plastic pollution environmental samples, screening of strains capable of utilizing PET as the sole carbon source has become an important way to obtain PET-degrading microbial resources. Although rapid progress has been made in the discovery and modification of PET hydrolases, most of them are focused on the biodegradation of pretreated PET or low-crystallinity PET, and there are still problems such as insufficient efficiency and limited stability in degrading high-crystallinity PET microplastics, which has become a key bottleneck restricting its practical application and industrialization.

[0005] In summary, in the prior art, high-crystallinity polyethylene terephthalate microplastics (hcPET-MPs) are difficult to be rapidly degraded in the natural environment due to the dense crystal region and limited effective exposed surface area, and long-term accumulation can easily cause persistent pollution. Traditional physical / chemical treatment generally has low efficiency, high cost and potential secondary pollution; and in the existing biodegradation system, the number of functional strains that can stably act on hcPET-MPs under neutral and moderate temperature conditions is limited, the overall degradation level reported is low, and many systems often need more harsh conditions or pretreatment to obtain observable results, and the characterization of material surface erosion process and evidence chain is not intuitive and systematic.

[0006] Based on the above problems, there is an urgent need for a method for screening new PET-degrading potential strains directly from marine plastic pollution samples. This method should be applicable to natural samples, have good repeatability, and be able to efficiently enrich and identify target strains with PET-degrading ability. The present invention is based on this demand, by using high-crystallinity PET microplastics (hcPET-MPs) as the sole carbon source for step-by-step enrichment, using inorganic salt-based medium for functional screening, and combining molecular biology and phylogenetic methods for identification, the marine-derived Pristinamycete Jou-S14 was successfully obtained, and its morphology, physiology, biochemistry and genetic characteristics were comprehensively described, providing a standardized, scientific and practical screening and identification process for the study of marine plastic-degrading microorganisms.

[0007] Therefore, the environmental pollution problem of hcPET-MPs is increasingly serious, and the traditional treatment method has many limitations. Microbial degradation as a green and sustainable technology provides a new direction for solving this problem. Based on the screening of Priestia aryabhattai Jou-S14 from the coastal area of Lianyungang City in Jiangsu Province, the high-efficiency degradation ability of hcPET-MPs is confirmed. These research results not only provide a theoretical basis for the biodegradation of hcPET-MPs, but also lay a practical foundation for the development of efficient plastic degradation technology. In the future, with the in-depth study of the microbial degradation mechanism and the optimization of technology, microbial degradation is expected to become one of the important means to solve the problem of plastic pollution. SUMMARY

[0008] Based on the problems and purposes mentioned in the background art, the present application proposes a Priestia aryabhattai Jou-S14 and its application. The strain has a weight loss rate of 5.02 ± 0.21% and 7.09 ± 0.35% for hcPET-MPs and high-crystallinity PET film within 30 days, respectively, and shows a weight loss effect of 1-2% for PA-MPs, PP-MPs, HDPE-MPs and PS-MPs and other microplastics. The results of SEM, FT-IR, XRD and LC-MS show that the strain Jou-S14 induces crystal zone damage and generates degradation products such as BHET and TPA through biofilm attachment, confirming the high-efficiency degradation ability of the strain for high-crystallinity PET microplastics. The present application first screens and identifies a new type of Priestia aryabhattai Jou-S14 strain from the coastal environment of Jiangsu. The strain Jou-S14 can utilize a variety of carbon sources, and solves the problems of low degradation efficiency of high-crystallinity PET, easy long-term accumulation of microplastics and lack of efficient treatment strain sources, and provides ideas for designing artificial communities for efficient degradation of high-crystallinity PET microplastics.

[0009] To achieve the above-mentioned purposes, the technical solutions adopted by the present application are as follows:

[0010] The first purpose of the present application is to propose a Priestia aryabhattai Jou-S14 strain, which has been preserved in the China General Microbiological Culture Collection Center on March 14, 2025, with the preservation number of CGMCC No.33815.

[0011] The second purpose of the present application is to propose the application of a Priestia aryabhattai Jou-S14 strain or its bacterial liquid in degrading microplastics and their films.

[0012] Further, the microplastics and the films thereof include one of polyethylene terephthalate (PET) microplastics, polyamide microplastics (PA-MPs), polypropylene microplastics (PP-MPs), high-density polyethylene microplastics (HDPE-MPs), and polystyrene microplastics (PS-MPs).

[0013] A third object of the present application is to provide a method for degrading polyethylene terephthalate plastic films, the method being as follows: cutting the hcPET films into square pieces, first immersing each film piece in a 2% SDS solution and anhydrous ethanol for more than 4 hours, respectively, and then washing with sterile water for 3 times; then, using sterile filter paper to absorb the moisture on the surface of the plastic film on an ultraclean workbench, and sterilizing with an ultraviolet lamp, and storing the film pieces for 3 days after sterilization for standby; next, taking 200 μL of the bacteria liquid of Priestia aryabhattai Jou-S14 and coating it on the CFBAM solid culture medium, taking sterile water as a control group, and placing the treated PET plastic film in the center of the culture dish, and culturing at 30 ℃ for 30 days.

[0014] Further, the LCFBM liquid medium is: 0.7 g of KH2PO4, 0.7 g of K2HPO4, 0.7 g of MgSO4·7H2O, 1.0 g of NH4NO3, 0.005 g of NaCl, 0.002 g of FeSO4·7H2O, 0.002 g of ZnSO4·7H2O, and 0.001 g of MnSO4·H2O.

[0015] The CFBAM solid culture medium is: adding 2% agar in the LCFBM, the LB liquid medium is: 10 g of tryptone, 10 g of sodium chloride, and 5 g of yeast extract, and the LB solid culture medium is: adding 2% agar in the LB liquid medium.

[0016] A fourth object of the present application is to provide a bacterial agent for degrading microplastics, the bacterial agent containing Priestia aryabhattai Jou-S14 as an active ingredient.

[0017] The above technical solution can achieve the following beneficial effects:

[0018] Compared with the prior art, the Jou-S14 strain can achieve higher and quantifiable weight loss degradation under milder conditions (the weight loss rates of hcPET-MPs and high crystallinity PET film within 30 days are 5.02 ± 0.21% and 7.09 ± 0.35%, respectively), and presents a 1-2% weight loss effect on PA-MPs, PP-MPs, HDPE-MPs, PS-MPs, etc., embodying a certain broad spectrum; at the same time, it has the potential to utilize various carbon sources to maintain growth and degradation activity, and strong adaptation and tolerance to environmental fluctuations / relatively extreme conditions, and is more suitable for complex pollution scenarios. The results of SEM, FT-IR, XRD and LC-MS show that the P. algi strain Jou-S14 can form a dense biofilm on the surface of hcPET-MPs and strongly adhere, resulting in surface roughening and the appearance of grooves / pits; FT-IR shows that the -OH peak is enhanced and the ester bond C=O and C-O peaks are weakened, confirming the main chain breakage; XRD shows that the crystallinity decreases and the crystal region order structure is disturbed; LC-MS detects products such as MHET and TPA, indicating that it degrades hcPET-MPs by hydrolysis accompanied by oxidation. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 is the weight loss rate of the Jou-S14 strain degrading different plastics.

[0020] Figure 2 is the colony morphology of the Jou-S14 strain after 10 days of growth in CFBAM medium with different types of microplastics.

[0021] Figure 3 is the morphology of the Jou-S14 strain in LCFBM containing 2% hcPET-MPs for 30 days. (Left: control group; right: Jou-S14 group)

[0022] Figure 4 is (A) the colony morphology of the Jou-S14 strain after 10 days of growth in CFBAM containing hcPET film; (B) SEM observation of the colonization of the Jou-S14 strain after 10 days of incubation on hcPET film (increase the magnification of the given area, as indicated by the rectangle); (C) the morphology of hcPET film treated with medium for 30 days (control); (D) SEM observation of hcPET film treated with medium for 30 days; (E) the morphology of hcPET film treated with the Jou-S14 strain for 30 days; (F) SEM observation of the degradation effect of hcPET film after 30 days of incubation with the Jou-S14 strain (magnify the given area, as indicated by the rectangle)

[0023] Figure 5Figure 1 is the colony morphology of strain Jou-S14 on LB medium (A) and CBFAM medium containing 2% hcPET-MPs (B), and the SEM observation of strain Jou-S14 (C). The phylogenetic tree based on 16S rDNA sequence is shown in (D), and * represents the 16S rDNA of strain Jou-S14.

[0024] Figure 6 Figure 2 is the growth curve of strain Jou-S14 at pH 5.0-9.0 (A), at 25-40 °C (B), and at different inoculation densities (OD 600 =0.1-0.5) (C), and the weight loss of hcPET-MPs degraded by strain Jou-S14 at various initial hcPET-MP concentrations (1-4%) for 30 days (D).

[0025] Figure 7 Figure 3 is the weight loss rate of hcPET-MPs degraded by strain Jou-S14 for 15-90 days (A), the pH change of hcPET-MPs degraded by strain Jou-S14 for 15-90 days (B), the growth curve of strain Jou-S14 in 2% hcPET-MPs as the sole carbon source for 8 days (C), and the growth curve of strain Jou-S14 in LCFBM medium with different carbon sources for 54 hours (D).

[0026] Figure 8 Figure 4 is the growth curve of strain Jou-S14 in LB medium for 54 hours.

[0027] Figure 9 Figure 5 is the SEM image of high crystallinity PET microplastics treated by strain Jou-S14 for 30 days.

[0028] Figure 10 Figure 6 is the FT-IR image of high crystallinity PET microplastics treated by strain Jou-S14 for 30 days.

[0029] Figure 11 Figure 7 is the XRD image of high crystallinity PET microplastics treated by strain Jou-S14 for 30 days.

[0030] Figure 12 Figure 8 is the LC-MS image of high crystallinity PET microplastics treated by strain Jou-S14 for 30 days. DETAILED DESCRIPTION

[0031] The present application will be further described below in conjunction with the accompanying drawings and examples. Figures 1-12 and examples.

[0032] The present embodiment gives a strain of Priestia aryabhattai Jou-S14, which has been deposited with the China General Microbiological Culture Collection Center on March 14, 2025, and has the accession number CGMCC No. 33815. The following is a process of screening, identification, and evaluation of the strain.

[0033] (1) Materials and methods:

[0034] hcPET-MPs (crystallinity 52 ± 3%, melting point 250 ± 5 ℃, average molecular weight 50,000 Da, density in the range of 1.401-1.446 g / cm 3 , hcPET film (crystallinity 37%, density 1.42 g / cm 3 , average molecular weight 30,000 Da, thickness about 3 mm) were purchased from China Huacheng Plastic Co., Ltd. PA-MPs, PP-MPs, HDPE-MPs and PS-MPs. LCFBM liquid medium: 0.7 g KH2PO4, 0.7 g K2HPO4, 0.7 g MgSO4·7H2O, 1.0 g NH4NO3, 0.005 g NaCl, 0.002 g FeSO4·7H2O, 0.002 g ZnSO4·7H2O, 0.001 g MnSO4·H2O. CFBAM solid medium: 2% agar was added to LCFBM. LB liquid medium: 10 g tryptone, 10 g sodium chloride, 5 g yeast extract, LB solid medium: 2% agar was added to LB liquid medium.

[0035] (2) Screening, identification and weight loss rate determination of hcPET-MPs degrading strains

[0036] Seawater and sea mud samples were collected from the coastal plastic pollution area of Lianyungang City, Jiangsu Province, and inoculated with 50 mL LCFBM medium containing 2% (m / v) hcPET-MPs. The enrichment liquid was gradient diluted and coated on CFBAM medium containing 2% hcPET-MPs, and cultured at 30 ℃ for 5-7 d. After repeated analysis and purification into single colonies, the degradation rate of the strains on hcPET-MPs was determined.

[0037] Single colonies were picked and inoculated onto LB medium and cultured at 30 °C and 180 rpm for 2 days. The bacterial cells were collected (5000 rpm, 5 min) and washed at least 3 times in LCFBM medium to adjust the OD. 600 To a concentration of 0.2-0.4. Add 2% sterile hcPET-MPs (sole carbon source) to the culture medium and incubate at 30℃ and 180 rpm for 30 days. The control group only received sterile water. Under these conditions, the degradation of hcPET films was measured. After incubation, the samples were filtered, washed with 2% (w / v) SDS to remove attached bacteria, rinsed with anhydrous ethanol, and the hcPET-MPs were recovered. The samples were dried at 60℃ to constant weight. The weight loss rate was calculated using the following formula:

[0038]

[0039] Where IWP is the initial weight and FWP is the final weight.

[0040] Using the genome of the hcPET-MPs degrading strain as a template, the bacterial 16S rRNA gene was amplified using universal primers 27F (5'-AGAGTTTGATCMTGGCTCAG-3') and 1492R (5'-TACGGYTACCTTGTTACGACTT-3'). After purification of the PCR target band, the sample was sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing analysis. The sequencing results were submitted to the NCBI database to obtain the GenBank accession number, and 16S rDNA phylogenetic analysis was performed using MEGA 11.0.

[0041] (3) Degradation evaluation of other microplastics and hcPET films

[0042] To visually observe the growth of strain Jou-S14 during the degradation of hcPET-MPs, this invention also explored the degradation of hcPET films. Three degradation methods were employed. The specific steps of the first method are as follows: hcPET films were cut into square fragments. Each fragment was first soaked in 2% SDS solution and anhydrous ethanol for more than 4 hours, followed by rinsing three times with sterile water. Then, the surface moisture of the plastic film was blotted dry with sterile filter paper on a laminar flow hood, and sterilized with ultraviolet light. After sterilization, the fragments were stored for 3 days for later use. Next, 200 μL of bacterial suspension was spread on CFBAM solid medium, with sterile water as a control group. The treated PET plastic film was placed in the center of a petri dish and cultured at 30 ℃ for 30 days.

[0043] The second method is to pick single colony from LB medium and perform three-zone streaking, then inoculate it into CFBAM solid medium containing 2% hcPET-MPs and incubate at 30°C for 10 days, and observe the growth of the colony. Based on this, the third degradation experiment is performed using hcPET film instead of microplastics. Finally, the weight loss rate of hcPET-MPs and hcPET film is measured to evaluate the weight loss rate.

[0044] To further explore the potential of Pristinamycetinresistens Jou-S14 in degrading other types of microplastics, the present application also designs experiments to use polyamide (PA), polypropylene (PP), high-density polyethylene (HDPE) and polystyrene (PS) as the sole carbon source to preliminarily evaluate the degradation effect of Jou-S14 strain on these different types of microplastics. The experimental method is the same as described in (2) above.

[0045] (4) Growth characteristics of strain Jou-S14

[0046] To explore the optimal biodegradation conditions of Jou-S14 strain in the process of hcPET-MPs degradation, the present application measures the optimal growth temperature and pH value of Jou-S14 strain. The specific experimental method is as follows: every 6 hours, 300 μL of Jou-S14 strain LB culture liquid is inoculated into a 96-well plate and cultured on a clean bench, and the optical density (OD600) is measured using a full-wavelength enzyme marker. The measured OD 600 value is taken as the vertical coordinate, and the culture time is taken as the horizontal coordinate, to draw the growth curve of Jou-S14 strain under different temperature and pH conditions.

[0047] Subsequently, the Jou-S14 strain cultured in LB medium is centrifuged at 5000 rpm for 5 minutes to remove the supernatant, and the precipitated bacterial body is resuspended in LCFBM medium. This centrifugation and resuspension step is repeated at least three times to ensure complete removal of residual LB medium. Subsequently, the recovered bacterial suspension is adjusted to OD 600For 0.2-0.4, and 1 g of hcPET-MPs was added. Incubation was carried out at 180 rpm and 30°C, and the OD value was measured at a wavelength of 600 nm to verify the survival and proliferation of the Jou-S14 strain during the degradation process. To dynamically evaluate the degradation effect of the Jou-S14 strain, the weight loss rate of the hcPET-MPs was regularly monitored every 15 days, and a dynamic change curve of the degradation efficiency was plotted with time (0-90 days) as the horizontal coordinate and the weight loss rate (%) as the vertical coordinate. At the same time, the pH change of the culture medium was measured every 15 days. The measured OD 600 value reflects the proliferation of microorganisms in the LCFBM medium, while the change in pH value reflects the metabolic activity of microorganisms. In LB medium, 30°C was incubated for 54 h, and OD 600 values were measured every 6 h and the growth curve of the Jou-S14 strain was plotted. In addition, the growth pattern of the Jou-S14 strain under other common carbon sources (xylitol, chitin, chitosan, glycerol, glucose, sucrose, carboxymethylcellulose sodium, lactose, sodium alginate, and LCFBM) was analyzed.

[0048] (5) Scanning electron microscope (SEM) observation

[0049] To verify the degradation effect and adhesion of the strain Jou-S14 on the surface of hcPET-MPs, the above samples were treated with 2% SDS solution for more than 12 hours to remove the surface bacterial cells, and then sequentially washed with anhydrous ethanol and distilled water, and dried at 60°C to constant weight before microstructure observation. To analyze the biofilm adhesion structure, the degraded samples were naturally air-dried, fixed on carbon tape, ion sputtered for 60 s, and then imaged using a scanning electron microscope (SEM) at an accelerating voltage of 15 kV.

[0050] (6) Fourier transform infrared spectroscopy (FT-IR) analysis

[0051] FT-IR was used to detect the chemical bond changes of the samples treated by the strain Jou-S14. The above samples were mixed with pure KBr at a mass ratio of 1:100, ground and pressed into a tablet, scanned at a wave number range of 400-4000 cm⁻¹ and a resolution of 4 cm⁻¹, and compared with the control group for functional group changes and new peak appearance

[0052] (7) X-ray diffraction (XRD) analysis

[0053] For the analysis of crystallinity changes, XRD measurements were performed using Cu Kα radiation (λ = 1.5418 Å). The samples were recorded in the range of 2θ = 5°-45°, with a scan rate of 1° / min, an electric current of 30 mA, a voltage of 40 kV, and measurements were performed at room temperature under constant conditions.

[0054] (8) Liquid chromatography-mass spectrometry (LC-MS) analysis

[0055] To identify the intermediates in the culture supernatant of Jou-S14 strain after 30 days of degradation of high crystallinity PET microplastics (hcPET-MPs), the culture solution was centrifuged to collect the supernatant, filtered through a 0.22 μm filter membrane, and then extracted with ethyl acetate, followed by analysis. The chromatographic conditions were as follows: a reversed-phase C18 chromatographic column (150 mm x 2.1 mm, 1.7 μm) was used, the mobile phase was water (0.1% formic acid) and acetonitrile (0.1% formic acid), and the flow rate was 0.3 mL·min⁻¹. The gradient elution program was as follows: the initial acetonitrile concentration was 5%, which was increased to 95% within 15 min and maintained for 3 min. The injection volume was 5 μL, and the column temperature was 40 ℃. The electrospray ionization (ESI) source temperature was set to 350℃, and the capillary voltage was 3.5 kV (positive ion mode). The mass spectrometer was operated in full scan mode, with a scan range of m / z 50-1000 amu, a resolution of 30,000 FWHM, and an automatic gain control (AGC) target value of 1 x 10 6 .

[0056] Experimental results:

[0057] (1) Screening of hcPET-MPs degrading strains and determination of degradation rate

[0058] The screening results showed that a total of 13 strains had the ability to degrade hcPET-MPs, and the hcPET-MPs degrading strains covered multiple genera (such as Pristinamycetium, Lactobacillus plantarum, Rhodobacter, Massilia, Bacillus, and Microbacterium folium, etc.). The determination of the weight loss rate of these strains on hcPET-MPs showed that these strains had different degrees of degradation (about 1-5%) on hcPET-MPs, among which the degradation ability of Pristinamycetium Jou-S14 was the most significant, with a weight loss rate of 5.02 ± 0.21% on hcPET-MPs. In addition, the Jou-S14 strain also showed certain degradation ability (weight loss rate distributed in 1-2%) on a variety of common plastics (hcPET film, HDPE-MPs, PA-MPs, PP-MPs, and PS-MPs), showing strong potential for multi-substrate degradation (see Figure 1 .

[0059] Colony morphology of Jou-S14 strain after 10 days of growth in CFBAM medium with different microplastics (see Figure 2 ).

[0060] The fermentation results showed that the Jou-S14 group appeared obvious milky white suspension compared with the control group (clear state), which also indicated that the Jou-S14 strain could use hcPET-MPs as carbon source and grow and reproduce (see Figure 3 ). Figure 3 Morphology of Jou-S14 strain in LCFBM containing 2% hcPET-MPs for 30 days. (Left: control group; right: Jou-S14 group).

[0061] However, the degradation rate of Jou-S14 strain on hcPET film was 7.09 ± 0.35%, which was higher than that of hcPET-MPs. After 10 days of culture, a large number of milky white colonies appeared on the film covered area, while no colony grew in the uncovered area and the control group (see Figure 4 A). Compared with the original, the cell volume of Jou-S14 strain decreased and the morphology was more regular. Thick biofilm was formed, and the bacteria adhered closely to the surface of microplastics. Biofilm promoted the growth and metabolic activity of bacteria, and enhanced the adhesion of bacteria, which improved the degradation ability of enzymes on the surface of microplastics. SEM showed high-density adhesion and local damage on the surface of the film (see Figure 4 B); after sterilization, the surface of hcPET film remained intact and smooth compared with the control group (see Figure 4 C-D), and the surface of hcPET film in Jou-S14 group showed obvious damage and holes, and local magnification area showed cell residues and surface roughening characteristics, which further indicated that strain Jou-S14 adhered to the surface of the film and induced the degradation of polyester structure (see Figure 4 E-F). Therefore, the selected P. algi Jou-S14 was selected for further study.

[0062] Figure 4 (A) Colony morphology of Jou-S14 strain after 10 days of growth in CFBAM containing hcPET film; (B) SEM observation of the colonization of Jou-S14 strain on hcPET film after 10 days of incubation (increase the magnification of the given area, as indicated by the rectangle); (C) Morphology of hcPET film treated with culture medium for 30 days (control); (D) SEM observation of hcPET film treated with culture medium for 30 days; (E) Morphology of hcPET film treated with Jou-S14 strain for 30 days; (F) SEM observation of the degradation effect of Jou-S14 strain on hcPET film after 30 days of incubation (magnify the given area, as indicated by the rectangle).

[0063] (3) Morphological observation and molecular biological identification of Jou-S14 strain

[0064] The colony of Jou-S14 strain on CFBAM medium containing hcPET-MPs was round, milky white, smooth and slightly convex, with neat edges (see Fig. 2A-B). Under scanning electron microscope, the strain was regular cylindrical, 2.65 x 1.22 pm, with spore structure and no branch formation (see Fig. 2C). Figure 5 A-B), scanning electron microscope under the strain was regular cylindrical, 2.65 x 1.22 pm, with spore structure and no branch formation (see Figure 5 C).

[0065] Figure 5 D). Based on the above results, the strain Jou-S14 was identified as Priestia aryabhattai, and has been preserved in the China General Microbiological Culture Collection Center (Preservation Number CGMCC No. 33815).

[0066] (4) Growth characteristics of the strain Priestia aryabhattai Jou-S14

[0067] The research of the present application shows that under the condition of pH 7.0 and 30°C, the growth of the strain Priestia aryabhattai Jou-S14 is the most vigorous, and the growth rate is significantly higher than that under other conditions (see Figure 6 A-B). When the initial OD 600 is about 0.2, the growth of the strain is the most stable, and the degradation efficiency is the highest (see Figure 6 C). In addition, the strain Jou-S14 can grow well under the condition of pH 5.0-9.0, and the OD 600 value can reach 1.0 under the temperature of 25-40°C, showing good environmental adaptability.

[0068] The strain Jou-S14 not only can efficiently degrade hcPET-MPs under neutral and mild conditions, but also can maintain the degradation activity in non-ideal environments such as low nutrition, pH fluctuation or temperature deviation. Further research shows that the concentration of 2% of hcPET-MPs is the optimal degradation condition, and under this concentration, the weight loss rate is close to 6% (see FIG. 6D). Higher or lower substrate concentration will inhibit the growth of the strain and the degradation activity, indicating that there is a balance between the surface site availability and enzyme adsorption and reaction rate in the degradation process.

[0069] The present application further optimizes the degradation experiment of the strain Priestia aryabhattai Jou-S14 under the culture condition of 2% hcPET-MPs as the only carbon source for 90 days. The results show that under the optimal growth condition, the degradation activity of the strain Jou-S14 is continuously enhanced, and the weight loss rate gradually increases (see Figure 7 A), and at 90 days, the weight loss rate reaches 12.10 ±0.24%. At the same time, the pH of the culture solution slightly decreases from the initial pH 7.0 to 6.8, indicating that acidic products such as BHET, MHET and TPA are generated in the degradation process (see Figure 7 B).

[0070] Under this condition, the growth of Jou-S14 strain was monitored. The growth curve of 8 days in 2% hcPET-MPs showed that the strain proliferated rapidly in the first 3 days, and the OD600 value continued to rise, and then gradually decreased to 0.5, entering the stationary phase, indicating that the metabolic activity reached a peak in the early stage (see Figure 7 C). This result shows that the degradation of Jou-S14 strain is mainly concentrated in the initial erosion stage of the surface of hcPET-MPs. In this stage, the bacteria form biofilm by rapid adhesion, which significantly enhances the interface contact between enzymes and substrates, thereby accelerating the occurrence of hydrolysis reaction.

[0071] This phenomenon shows that when hcPET-MPs are used as a carbon source, due to its dense structure, the released small molecules cannot meet the growth needs of the strain, resulting in slow growth and decreased biomass.

[0072] Further analysis of the growth of Jou-S14 strain in other common carbon sources (such as glucose, sucrose, glycerol and chitin, etc.) (see Figure 7 D). The results show that the strain can grow well in various carbon sources, especially in chitin, glucose and chitosan systems, showing its broad-spectrum carbon source adaptability. This characteristic may be due to the ability of Jou-S14 strain to express multiple glycoside hydrolase and lipase family members, providing diverse enzymatic reaction pathways, thereby enhancing its feasibility and degradation efficiency in the recycling of various plastic waste.

[0073] In addition, the growth curve of strain Jou-S14 in LB medium was also determined, and the results showed that its highest OD600 value can reach 1.8, and then enters the stationary phase (see Figure 8).

[0074] (5) Scanning electron microscope (SEM) results of high crystallinity PET microplastics (hcPET-MPs) after degradation

[0075] Scanning electron microscope (SEM) is a high-efficiency preliminary identification method for microplastic degradation. By observing SEM images, the formation and adhesion of bacterial biofilm and the changes in the structure of plastic surfaces can be clearly revealed. High crystallinity PET microplastics treated by Jou-S14 strain were analyzed by SEM, and the microbial growth of Jou-S14 strain on the surface of high crystallinity PET microplastics was observed. The results are as follows Figure 9As shown, the pre-degradation showed a smoother surface compared with the degraded high crystallinity PET microplastics, while the surface of the degraded was rough. The surface of the polymer structure of the sample lost smoothness after the degradation process, and the present application used SEM technology to observe the surface characteristics of PET microplastics and the formation of biofilm after 30 days of Jou-S14 microbial community treatment. Jou-S14 strain formed a large number of biofilms attached to or embedded in the interior of high crystallinity PET microplastics, and had strong adhesion. The surface of high crystallinity PET microplastics treated by Jou-S14 strain showed obvious damage, and the local area was uneven, showing gullies, grooves and holes, and the damage degree was obviously higher than that before degradation. In summary, the above results show that Jou-S14 strain can efficiently degrade hcPET-MPs.

[0076] (6) FT-IR results of high crystallinity PET microplastics (hcPET-MPs) after degradation

[0077] Fourier transform infrared spectroscopy (FT-IR) is an efficient preliminary identification method for microplastics degradation. By comparing the changes of characteristic absorption peaks of samples before and after degradation, the generation and transformation of plastic surface functional groups and the destruction of polyester main chain structure can be directly reflected. The present application analyzed the high crystallinity PET microplastics (hcPET-MPs) treated by Jou-S14 strain by FT-IR, and the results are shown in Figure 10 As shown, after treatment, an obvious -OH stretching vibration peak appeared at 3430 cm⁻¹, indicating that hydroxyl-containing compounds were generated during the degradation process, and the material surface properties gradually changed from hydrophobic to hydrophilic. At the same time, the ester group C=O absorption peak at 1717 cm⁻¹ was significantly weakened, indicating that the ester bond in the polyester was broken. And the C-O stretching vibration peak at 1265 cm⁻¹ was also reduced, further confirming that the polyester main chain structure was destroyed. In addition, the aromatic ring C=C vibration peak at 1640 cm⁻¹ was enhanced, indicating the formation of TPA and other aromatic hydrolysis products. In summary, the systematic changes of FT-IR characteristic peaks prove that Jou-S14 strain can effectively promote the hydrolytic degradation process of hcPET-MPs from the functional group level.

[0078] (7) XRD results of high crystallinity PET microplastics (hcPET-MPs) after degradation

[0079] X-ray diffraction (XRD) is a high-efficiency method for characterizing the change of crystalline structure of microplastics and for preliminary identification of degradation effect. By comparing the diffraction peak intensity, peak shape and the appearance of characteristic diffraction peaks of the sample before and after degradation, the change of polymer crystallinity, the destruction of ordered segment accumulation structure and the generation of degradation products can be clearly revealed. The present application analyzes the XRD of high crystallinity PET microplastics (hcPET-MPs) before and after treatment by Jou-S14 strain, and the results are shown in Figure 11 As compared with the sample before treatment, the main diffraction peak intensity of the sample after treatment is significantly reduced, and the peak shape appears broadening, indicating that the crystallinity of hcPET-MPs is significantly decreased and the order of crystal region is weakened. At the same time, the characteristic diffraction peaks corresponding to degradation products such as BHET, MHET and TPA can be detected in the diffraction pattern, indicating that the corresponding small molecule / monomer products have been generated in the degradation process. In summary, the XRD results prove that Jou-S14 strain can act on the amorphous region and further disturb the ordered accumulation structure of polymer chain, thereby promoting the degradation of hcPET-MPs from the crystalline structure level.

[0080] (8) LC-MS results of hcPET-MPs after degradation

[0081] Liquid chromatography-mass spectrometry (LC-MS) is a high-efficiency method for identifying microplastic degradation products. By qualitatively analyzing the small molecule products in the culture supernatant, the generation of intermediates and end products in the polyester hydrolysis and subsequent oxidative conversion process can be clearly revealed. The present application detects the LC-MS of the hcPET-MPs fermentation broth treated by Jou-S14 strain, and the results are shown in Figure 12 A variety of products related to PET degradation are detected in the fermentation broth, including TPA, MHET, phenylacetaldehyde, 2,3-dihydro-1-benzofuran-2-carboxylic acid, diacetyl m-benzotriol and diphenic acid diglycol ester, etc. The above detection results show that Jou-S14 strain can gradually cleave the polyester backbone through hydrolysis and further convert the degradation products through oxidative reaction, thereby generating MHET, TPA and other aromatic compounds; at the same time, the appearance of intermediates such as phenylacetaldehyde and diacetyl m-benzotriol further confirms that the aromatic ring structure continuously breaks and transforms in the degradation process. In summary, it is shown that Jou-S14 strain can effectively degrade hcPET-MPs and has significant degradation ability.

[0082] The above-mentioned are preferred embodiments of the present application. For those skilled in the art, various equivalent modifications of the present application without departing from the principles of the present application are within the protection scope of the appended claims of the present application.

Claims

1. A type of Priestia aryabhattai (Jou-S14), characterized in that: The bacterium *Priestia aryabhattai*, Jou-S14, was deposited on March 14, 2025, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 33815.

2. The application of *Priestia aryabhattai* Jou-S14 or its bacterial suspension as described in claim 1 in the degradation of microplastics and their films, characterized in that: Microplastics and their films include one of the following: polyethylene terephthalate (PET) microplastics, polyamide microplastics (PA-MPs), polypropylene microplastics (PP-MPs), high-density polyethylene microplastics (HDPE-MPs), and polystyrene microplastics (PS-MPs).

3. A method for degrading polyethylene terephthalate plastic film, characterized in that: The method is as follows: hcPET film is cut into square segments. Each segment is first soaked in 2% SDS solution and anhydrous ethanol for more than 4 hours, then rinsed 3 times with sterile water. Next, the surface moisture of the plastic film is blotted dry with sterile filter paper on a clean bench, and then sterilized with ultraviolet light. After sterilization, the film is stored for 3 days for later use. Then, 200 μL of *Priestia aryabhattai* Jou-S14 bacterial suspension as described in claim 1 is spread on CFBAM solid medium. Sterile water is used as a control group. The treated PET plastic film is placed in the center of a petri dish and cultured at 30 ℃ for 30 days. The CFBAM liquid medium consists of: 0.7 g KH2PO4, 0.7 g K2HPO4, 0.7 g MgSO4·7H2O, 1.0 g NH4NO3, 0.005 g NaCl, 0.002 g FeSO4·7H2O, 0.002 g MgSO4·7H2O, 0.002 ... g ZnSO4·7H2O, 0.001 g MnSO4·H2O; CFBAM solid medium: 2% agar added to LCFBM; LB liquid medium: 10 g tryptone, 10 g sodium chloride, 5 g yeast extract; LB solid medium: 2% agar added to LB liquid medium.

4. A microbial agent for degrading microplastics, characterized in that: The bacterial agent contains Priestia aryabhattai Jou-S14 as the active ingredient.

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