Prussella adamsii Jou-S14 and application thereof

Abstract

The strain is preserved in the China General Microbiological Culture Collection Center (CGMCC) on March 14, 2025, and the preservation number is CGMCC No.33815. The weight loss ratios of the strain to hcPET-MPs and a high-crystallinity PET film within 30 days are 5.02 + / -0.21% and 7.09 + / -0.35% respectively, and the strain has the advantages that the strain is high in yield, high in yield and good in stability; and the composite material has a weight loss effect of 1-2% on a plurality of micro-plastics such as PA-MPs, PP-MPs, HDPE-MPs, PS-MPs and the like.

Description

Technical Field

[0001] This invention belongs to the field of environmental microbiology technology, specifically relating to a strain of Priestella auriculi Jou-S14 and its applications. Background Technology

[0002] With the continuous increase in global plastic production, polyethylene terephthalate (PET), as a commonly used thermoplastic polyester material, is widely used in packaging, bottled water, textile fibers, and the food industry. Large amounts of PET waste enter the natural environment, accumulating over time and gradually breaking down into PET microplastics (PET-MPs). Particularly in marine environments, due to their non-degradable nature and highly crystalline structure, PET microplastics maintain chemical stability over multi-year timescales, posing serious ecological and environmental risks. Numerous studies have shown that the highly crystalline structure of PET makes it difficult for enzymes or microorganisms to attack its internal ester bonds, becoming a major factor limiting microbial degradation efficiency. Therefore, microbial resources capable of directly utilizing environmental PET waste or PET microplastics as the sole carbon source for growth are extremely scarce, and the screening of relevant strains typically requires a complex and lengthy enrichment and rigorous screening process.

[0003] Current research has reported microorganisms and enzymes capable of degrading low-crystallinity PET films or artificially modified PET materials, such as PETase derived from Ideonella sakaiensis. However, these strains typically struggle to degrade naturally occurring PET waste with high crystallinity (>30%), and their degradation capabilities often depend on PET pretreatment, such as heat treatment, surface abrasion, or solvent expansion. Therefore, directly screening natural strains adapted to complex conditions such as high salinity, temperature fluctuations, ultraviolet radiation, and the coexistence of multiple plastics, and possessing the potential to degrade high-crystallinity PET, is of significant scientific and applied importance for the remediation of microplastic pollution and the discovery of novel ester hydrolases.

[0004] Coastal sediments and seawater-plastic interface biofilms are important natural resources for plastic-degrading microorganisms. PET waste exposed to the marine environment for extended periods often develops complex microbial colonization systems on its surface, containing a large number of potentially functional bacteria with oxidoreductase, ester hydrolase, and aromatic compound metabolic capabilities. Therefore, targeted enrichment of marine plastic-polluted environmental samples and screening for strains capable of utilizing PET as the sole carbon source have become crucial pathways for obtaining PET-degrading microbial resources. Although rapid progress has been made in the discovery and modification of PET hydrolases, most studies focus on the pre-treated PET or the biodegradation of low-crystallinity PET. In the degradation of high-crystallinity PET microplastics, insufficient efficiency and limited stability remain, becoming key bottlenecks restricting their practical application and industrialization.

[0005] In summary, in existing technologies, highly crystalline polyethylene terephthalate microplastics (hcPET-MPs) are difficult to degrade rapidly in the natural environment due to their dense crystalline regions and limited effective exposed surface area, leading to persistent pollution over long periods. Traditional physical / chemical treatments generally suffer from low efficiency, high cost, and potential secondary pollution. Furthermore, existing biodegradation systems have a limited number of functional bacterial strains that can stably act on hcPET-MPs under neutral and moderate-temperature conditions, and the overall reported degradation levels are generally low. Many systems often require more stringent conditions or pretreatment to achieve significant results, and the characterization of the material surface erosion process and the chain of evidence is not intuitive or systematic enough.

[0006] Based on the aforementioned problems, there is an urgent need for a method to directly screen novel PET-degrading strains from marine plastic-polluted samples. This method should be applicable to natural samples, have good reproducibility, and be able to efficiently enrich and identify target strains with PET-degrading capabilities. This invention addresses this need by using highly crystalline PET microplastics (hcPET-MPs) as the sole carbon source for stepwise enrichment, employing inorganic salt basal culture media for functional screening, and combining molecular biology and phylogenetic methods for identification. This successfully yielded marine-derived *Priscilla auriculi* Jou-S14, and its morphological, physiological, biochemical, and genetic characteristics were comprehensively elucidated. This provides a standardized, scientific, and practical screening and identification process for the study of marine plastic-degrading microorganisms.

[0007] Therefore, the environmental pollution problem caused by hcPET-MPs is becoming increasingly serious, and traditional treatment methods have many limitations. Microbial degradation, as a green and sustainable technology, provides a new direction for solving this problem. This invention, based on *Priscilla auriculi* Jou-S14 screened from the coastal area of ​​Lianyungang City, Jiangsu Province, demonstrates its highly efficient degradation ability of hcPET-MPs. 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 technologies. In the future, with in-depth research on the microbial degradation mechanism and technological optimization, microbial degradation is expected to become one of the important means to solve the plastic pollution problem. Summary of the Invention

[0008] Based on the problems and objectives mentioned in the background art, this invention proposes a strain of *Priscilla argentea*, Jou-S14, and its applications. This strain exhibits weight loss rates of 5.02 ± 0.21% for hcPET-MPs and 7.09 ± 0.35% for highly crystalline PET films within 30 days, and shows a weight loss effect of 1-2% for various microplastics such as PA-MPs, PP-MPs, HDPE-MPs, and PS-MPs. Combined results from SEM, FT-IR, XRD, and LC-MS indicate that strain Jou-S14 induces crystalline region destruction and generates degradation products such as BHET and TPA through biofilm adhesion, confirming the strain's ability to efficiently degrade highly crystalline PET microplastics. This invention is the first to screen and identify a novel strain of Priestella auriculi, Jou-S14, from the coastal environment of Jiangsu Province. This strain can utilize multiple carbon sources and solves the problems of low degradation efficiency of highly crystalline PET, easy long-term accumulation of microplastics, and lack of efficient treatment strain sources. It also provides ideas for designing artificial communities that can efficiently degrade highly crystalline PET microplastics.

[0009] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0010] The first objective of this invention is to provide a strain of *Priestia aryabhattai*, Jou-S14, which was deposited on March 14, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 33815.

[0011] The second objective of this invention is to propose the application of Priestia aryabhattai Jou-S14 or its bacterial culture in the degradation of microplastics and their films.

[0012] Furthermore, 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).

[0013] The third objective of this invention is to provide a method for degrading polyethylene terephthalate (PET) plastic film, which 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 three 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 segments are stored for 3 days for later use. Then, 200 μL of *Priestia aryabhattai* Jou-S14 bacterial suspension is spread on CFBAM solid medium, with sterile water 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.

[0014] Furthermore, the LCFBM liquid medium contains: 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, and 0.001 g MnSO4·H2O.

[0015] 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.

[0016] The fourth objective of this invention is to provide a microbial agent for degrading microplastics, wherein the microbial agent contains Priestia aryabhattai Jou-S14 as an active ingredient.

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

[0018] Compared with existing technologies, the Jou-S14 strain can achieve higher and quantifiable weight loss degradation under milder conditions (weight loss rates of 5.02 ± 0.21% and 7.09 ± 0.35% for hcPET-MPs and high-crystallinity PET films within 30 days, respectively), and exhibits a weight loss effect of 1-2% on PA-MPs, PP-MPs, HDPE-MPs, and PS-MPs, demonstrating a certain broad spectrum. At the same time, it has the potential to maintain growth and degradation activity by utilizing multiple carbon sources and to adapt to and tolerate environmental fluctuations / relatively extreme conditions, making it more suitable for complex pollution scenarios. The combined results of SEM, FT-IR, XRD, and LC-MS showed that *Primatellella asiatica* Jou-S14 could form a dense biofilm on the surface of hcPET-MPs and adhere strongly, leading to surface roughening and the appearance of grooves / pits. FT-IR showed an enhanced –OH peak and weakened C=O and CO peaks of the ester bonds, confirming main chain breakage. XRD indicated decreased crystallinity and disturbance of the ordered structure of the crystalline regions. LC-MS detected products such as MHET and TPA, indicating that it gradually degrades hcPET-MPs through hydrolysis accompanied by oxidation. Attached Figure Description

[0019] Figure 1 This represents the weight loss rate of different plastics degraded by the Jou-S14 strain.

[0020] Figure 2 The colony morphology of strain Jou-S14 after 10 days of growth on CFBAM medium containing different types of microplastics.

[0021] Figure 3 The morphology of strain Jou-S14 after 30 days in LCFBM containing 2% hcPET-MPs. (Left: Control group; Right: Jou-S14 group)

[0022] Figure 4 (A) Colony morphology of strain Jou-S14 grown in CFBAM containing hcPET film after 10 days; (B) SEM observation of colonization of strain Jou-S14 on hcPET film after 10 days of incubation (increase the magnification of the given area, as shown 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 strain Jou-S14 for 30 days; (F) SEM observation of degradation effect of strain Jou-S14 on hcPET film after 30 days of incubation (magnify the given area, as shown by the rectangle).

[0023] Figure 5(A) Colony morphology of strain Jou-S14 on LB medium; (B) Colony morphology of strain Jou-S14 on CBFAM medium containing 2% hcPET-MPs; (C) Morphology of strain Jou-S14 observed by SEM; (D) Phylogenetic tree constructed based on 16S rDNA sequence, * represents 16S rDNA of strain Jou-S14.

[0024] Figure 6 (A) Growth curve of strain Jou-S14 at pH 5.0–9.0; (B) Growth curve of strain Jou-S14 at 25–40 °C; (C) Growth curve of strain Jou-S14 at different inoculation densities (OD). 600 (D) hcPET-MPs degraded by strain Jou-S14 showed a 30-day weight loss at various initial hcPET-MP concentrations (1-4%).

[0025] Figure 7 (A) Weight loss rate of strain Jou-S14 degrading hcPET-MPs from 15 to 90 days; (B) pH changes of strain Jou-S14 degrading hcPET-MPs from 15 to 90 days; (C) Growth curve of strain Jou-S14 in 2% hcPET-MPs (as the sole carbon source) for 8 days; (D) Growth curve of strain Jou-S14 in LCFBM medium with different carbon sources for 54 hours.

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

[0027] Figure 9 This is a SEM image of highly crystalline PET microplastics after being treated with the Jou-S14 strain for 30 days.

[0028] Figure 10 This is an FT-IR image of highly crystalline PET microplastics after being treated with the Jou-S14 strain for 30 days.

[0029] Figure 11 This is an XRD image of highly crystalline PET microplastics after being treated with the Jou-S14 strain for 30 days.

[0030] Figure 12 This is an LC-MS image of highly crystalline PET microplastics after being treated with the Jou-S14 strain for 30 days. Detailed Implementation

[0031] The following is in conjunction with the appendix Figure 1-12 The present invention will be further illustrated by the following examples:

[0032] This embodiment presents a strain of *Priestia aryabhattai*, Jou-S14, which was deposited on March 14, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 33815. The following describes the screening, identification, and evaluation process of this strain.

[0033] (1) Materials and methods:

[0034] hcPET-MPs (crystallinity 52±3%, melting point 250±5 ℃, average molecular weight 50,000 Da, density 1.401-1.446 g / cm³) 3 Within the specified range), hcPET film (crystallinity 37%, density 1.42 g / cm³). 3 The polyamide microplastics (PA-MPs), polypropylene microplastics (PP-MPs), high-density polyethylene microplastics (HDPE-MPs), and polystyrene microplastics (PS-MPs), with an average molecular weight of 30,000 Da and a thickness of approximately 3 mm, were all purchased from China Huachuang Plastics Co., Ltd. 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: LCFBM with 2% agar added. LB liquid medium: 10 g tryptone, 10 g sodium chloride, 5 g yeast extract. LB solid medium: LB liquid medium with 2% agar added.

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

[0036] Marine mud and seawater samples were collected from plastic-polluted coastal areas of Lianyungang City, Jiangsu Province. The samples were inoculated with 50 mL of LCFBM medium containing 2% (m / v) hcPET-MPs and directionally enriched at 30 ℃ and 180 rpm for 90 days (each 30-day cycle). The enriched solution was then serially diluted and spread onto CFBAM medium containing 2% hcPET-MPs, and incubated statically at 30 ℃ for 5–7 days. After repeated purification, the degradation rate of hcPET-MPs was determined by analyzing the purified single-cell fallouts.

[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 for 30 days at 30℃ and 180 rpm. The control group received only 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 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 involves picking a single colony from LB medium and streaking it in three zones, then inoculating it into CFBAM solid medium containing 2% hcPET-MPs and culturing it at 30 °C for 10 days to observe colony growth. Based on this, a third degradation experiment was conducted using hcPET film sheets instead of microplastics. Finally, the weight loss rates of hcPET-MPs and hcPET film were measured to evaluate their weight loss efficiency.

[0044] To further explore the potential of *Priscilla argentea* Jou-S14 in degrading other types of microplastics, this invention also designed an experiment using microplastics such as polyamide (PA), polypropylene (PP), high-density polyethylene (HDPE), and polystyrene (PS) as the sole carbon source to preliminarily evaluate the degradation effect of strain Jou-S14 on these different types of microplastics. The experimental method is the same as that described in (2) above.

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

[0046] To investigate the optimal biodegradation conditions of strain Jou-S14 during the degradation of hcPET-MPs, this study determined the optimal growth temperature and pH value of strain Jou-S14. The specific experimental method is as follows: Every 6 hours, 300 μL of LB broth of strain Jou-S14 was inoculated into 96-well plates, cultured on a clean bench, and its optical density (OD600) was measured using a full-wavelength microplate reader. The measured OD600 was used as the reference value. 600 The values ​​are plotted on the ordinate and the culture time on the abscissa to plot the growth curves of strain Jou-S14 under different temperature and pH conditions.

[0047] Subsequently, the Jou-S14 strain cultured in LB medium was centrifuged at 5000 rpm for 5 minutes to remove the supernatant, and the precipitated bacterial cells were resuspended in LCFBM medium. This centrifugation and resuspension step was repeated at least three times to ensure complete removal of residual LB medium. The recovered bacterial suspension was then adjusted to OD0.05. 600The concentration of the medium was 0.2-0.4, and 1 g of hcPET-MPs was added. The culture was carried out at 180 rpm and 30 ℃, and the OD value was measured at 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 hcPET-MPs was monitored every 15 days, and a dynamic change curve of degradation efficiency was plotted with time (0-90 days) as the x-axis and weight loss rate (%) as the y-axis. Simultaneously, the pH change of the culture medium was measured every 15 days. The measured OD values... 600 The pH value reflects the proliferation of microorganisms in LCFBM medium, while changes in pH reflect the metabolic activity of microorganisms. OD values ​​were measured every 6 hours after incubation in LB medium at 30 °C for 54 h. 600 The growth curve of strain Jou-S14 was plotted. Furthermore, the growth patterns of strain Jou-S14 under other common carbon sources (xylitol, chitin, chitosan, glycerol, glucose, sucrose, sodium carboxymethyl cellulose, lactose, sodium alginate, and LCFBM) were analyzed.

[0048] (5) Observation by scanning electron microscope (SEM)

[0049] To verify the degradation and adhesion of strain Jou-S14 on the surface of hcPET-MPs, the samples were treated with 2% SDS solution for more than 12 hours to remove surface bacterial cells, followed by sequential washing with anhydrous ethanol and distilled water, and then dried at 60 °C to constant weight for microstructural observation. To analyze the biofilm adhesion structure, the degraded samples were air-dried, fixed onto carbon tape, and ion-sputtered with gold for 60 s. Imaging was then performed 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 changes in chemical bonds in samples treated with strain Jou-S14. The samples were mixed with pure KBr at a mass ratio of 1:100, ground, and compressed into pellets. The pellets were scanned within the wavenumber range of 400-4000 cm⁻¹ and at a resolution of 4 cm⁻¹. Changes in functional groups and the appearance of new peaks were compared with a control group.

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

[0053] To analyze the changes in crystallinity, Cu Kα radiation (λ = 1.5418 Å) was used for XRD analysis. The diffraction patterns of the samples were recorded at 2θ = 5°–45°, with a scan rate of 1° / min, a current of 30 mA, and a voltage of 40 kV, under constant room temperature conditions.

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

[0055] Intermediate products were identified in the culture supernatant of strain Jou-S14 after 30 days of degradation of highly crystalline PET microplastics (hcPET-MPs). The culture medium was centrifuged to collect the supernatant, filtered through a 0.22 μm filter, and extracted with ethyl acetate before analysis. Chromatographic conditions: A reversed-phase C18 column (150 mm × 2.1 mm, 1.7 μm) was used. The mobile phase was water (0.1% formic acid) and acetonitrile (0.1% formic acid), with a flow rate of 0.3 mL·min⁻¹. The gradient elution program was: initial acetonitrile 5%, increased to 95% within 15 min, and held 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 and a resolution of 30,000 FWHM. The automatic gain control (AGC) target value was 1 × 10⁻⁶. 6 .

[0056] Experimental results:

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

[0058] Screening results showed that 13 strains were capable of degrading hcPET-MPs, covering multiple genera (such as *Priscilla auriculata*, *Lactobacillus plantarum*, *Rhodotorula*, *E. variegata*, *Bacillus*, and *Microbacterium leafii*). The weight loss rate of these strains on hcPET-MPs was measured, and the results showed that these strains degraded hcPET-MPs to varying degrees (approximately 1-5%), with *Priscilla auriculata* (Jou-S14) exhibiting the most significant degradation ability, reaching a weight loss rate of 5.02 ± 0.21%. Furthermore, strain Jou-S14 also showed some degradation ability on various common plastics (hcPET film, HDPE-MPs, PA-MPs, PP-MPs, and PS-MPs) (weight loss rates ranging from 1-2%), demonstrating its strong multi-substrate degradation potential (see...). Figure 1 ).

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

[0060] Fermentation results showed that, compared to the control group (clear state), the Jou-S14 group exhibited a distinct milky white turbidity, indicating that the Jou-S14 strain can utilize hcPET-MPs as a carbon source for growth and reproduction (see...). Figure 3 ). Figure 3 Morphology of strain Jou-S14 in LCFBM containing 2% hcPET-MPs for 30 days. (Left: control group; Right: Jou-S14 group).

[0061] However, the degradation rate of hcPET film by strain Jou-S14 was 7.09 ± 0.35%, which was higher than that of hcPET-MPs. When Jou-S14 bacterial culture was coated onto the surface of CFBAM and covered with hcPET film, after 10 days of incubation, a large number of milky-white colonies appeared in the covered area, while no colonies grew in the uncovered area and the control group (see...). Figure 4 A). Compared to the original strain, the Jou-S14 cells were smaller and more regularly shaped. A thick biofilm formed, with the bacteria tightly adhering to the microplastic surface. The biofilm promoted bacterial growth and metabolic activity, enhanced bacterial adhesion, and improved the degradation capacity of enzymes on the microplastic surface. SEM showed high-density adhesion and localized damage on the membrane surface (see [link]). Figure 4 B); After sterilization, the surface of the hcPET film remained intact and smooth compared to the control group (see...). Figure 4 (CD) The hcPET film surface of the Jou-S14 group showed obvious damage and pores. Local magnified areas revealed cell residue and surface roughening characteristics, further indicating that strain Jou-S14 attached to the film surface and induced the degradation of the polyester structure (see CD). Figure 4 Therefore, *Priscilla argentea* Jou-S14 was selected for further research.

[0062] Figure 4 (A) Colony morphology of the Jou-S14 strain grown in CFBAM containing hcPET film after 10 days; (B) SEM observation of the colonization of the Jou-S14 strain on hcPET film after 10 days of incubation (increase the magnification of the given area, as shown 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 the Jou-S14 strain on hcPET film after 30 days of incubation (magnify the given area, as shown by the rectangle).

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

[0064] The colonies of strain Jou-S14 on CFBAM medium containing hcPET-MPs were round, milky white, with a smooth, rounded, slightly raised surface and neat edges (see...). Figure 5 (AB) The strain under a scanning electron microscope appears as a regular cylindrical shape, 2.65 × 1.22 µm, with spore structure and no branching (see AB). Figure 5 C).

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

[0066] (4) Results of growth characteristics of *Priscilla argentea* Jou-S14

[0067] This invention demonstrates that strain *Priscilla argentea* Jou-S14 exhibits the most vigorous growth under conditions of pH 7.0 and 30 °C, with a growth rate significantly higher than under other conditions (see [link to study]). Figure 6 AB). When the initial OD 600 At a concentration of approximately 0.2, the strain exhibited the most stable growth and the highest degradation efficiency (see...). Figure 6 C). Furthermore, strain Jou-S14 grew well under conditions ranging from pH 5.0 to 9.0, and its OD value increased significantly at temperatures from 25 to 40 °C. 600 The value can still reach 1.0, showing good environmental adaptability.

[0068] The Jou-S14 strain not only efficiently degrades hcPET-MPs under neutral and mild conditions, but also maintains degradation activity in non-ideal environments such as low nutrition, pH fluctuations, or temperature deviations. Further studies showed that a 2% concentration of hcPET-MPs was the optimal degradation condition, at which the weight loss rate approached 6% (see Figure 6D). Higher or lower substrate concentrations inhibited cell growth and degradation activity, indicating a balance between surface site availability and enzyme adsorption and reaction rate during degradation.

[0069] This invention further optimized the degradation experiment of strain *Priscilla aspergillus* Jou-S14 under the culture condition of 2% hcPET-MPs as the sole carbon source for 90 days. The results showed that under optimal growth conditions, the degradation activity of strain Jou-S14 was continuously enhanced, and the weight loss rate gradually increased (see...). Figure 7 (A) By day 90, the weight loss reached 12.10 ± 0.24%. Simultaneously, the pH of the culture medium decreased slightly from the initial pH 7.0 to 6.8, indicating the formation of acidic products such as BHET, MHET, and TPA during degradation (see [link to culture medium]). Figure 7 B).

[0070] Under these conditions, the growth of strain Jou-S14 was monitored. The growth curve after 8 days of culture in 2% hcPET-MPs showed that the strain proliferated rapidly in the first 3 days, with the OD600 value continuously increasing, then gradually decreasing to 0.5, entering a stationary phase, indicating that metabolic activity reached its peak in the initial stage (see...). Figure 7 C). This result indicates that the degradation activity of strain Jou-S14 is mainly concentrated in the initial erosion stage on the surface of hcPET-MPs. During this stage, the bacteria rapidly attach and form a biofilm, significantly enhancing the interfacial contact between the enzyme and the substrate, thereby accelerating the hydrolysis reaction.

[0071] This phenomenon indicates that when hcPET-MPs are used as a carbon source, their dense structure prevents the release of small molecules from meeting the growth needs of the strain, resulting in slow growth and reduced biomass.

[0072] The growth patterns of strain Jou-S14 in other common carbon sources (such as glucose, sucrose, glycerol, and chitin) were further analyzed (see...). Figure 7 (D) The results showed that the strain could grow well under various carbon source conditions, especially in the chitin, glucose, and chitosan systems, demonstrating its broad-spectrum carbon source adaptability. This characteristic may stem from the fact that the Jou-S14 strain can provide diverse enzymatic reaction pathways by expressing multiple glycoside hydrolases and lipase family members, thereby enhancing its feasibility and degradation efficiency in the recycling of various plastic wastes.

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

[0074] (5) Scanning electron microscopy (SEM) results of highly crystalline PET microplastics (hcPET-MPs) after degradation

[0075] Scanning electron microscopy (SEM) is a highly efficient method for preliminary identification of microplastic degradation. By observing SEM images, the formation and attachment of bacterial biofilms and changes in the plastic surface structure can be clearly revealed. High-crystallinity PET microplastics treated with the Jou-S14 strain were analyzed using SEM, and the microbial growth of the Jou-S14 strain on the surface of the high-crystallinity PET microplastics was observed. The results are as follows: Figure 9As shown, compared with the degraded highly crystalline PET microplastics, the surface before degradation was smoother, while the surface after degradation was rough. The polymer structure surface of the sample lost its smoothness after the degradation process. Furthermore, this invention used SEM technology to observe the surface characteristics of PET microplastics and the formation of biofilms after 30 days of treatment with the Jou-S14 microbial community. The Jou-S14 strain formed a large number of biofilms attached to or embedded within the highly crystalline PET microplastics on the microplastics or plastic films, exhibiting strong adhesion. The surface of the highly crystalline PET microplastics treated with the Jou-S14 strain showed obvious damage, with unevenness in local areas, displaying grooves, depressions, and pits, and the degree of damage was significantly higher than that before degradation. In conclusion, the above results show that the Jou-S14 strain can efficiently degrade hcPET-MPs.

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

[0077] Fourier transform infrared spectroscopy (FT-IR) is a highly efficient method for preliminary identification of microplastic degradation. By comparing the changes in characteristic absorption peaks of samples before and after degradation, the formation and transformation of functional groups on the plastic surface and the damage to the polyester backbone structure can be directly reflected. This invention uses FT-IR analysis on highly crystalline PET microplastics (hcPET-MPs) before and after treatment with the Jou-S14 strain. The results are as follows: Figure 10 As shown, a distinct -OH stretching vibration peak appeared at 3430 cm⁻¹ after treatment, indicating the formation of hydroxyl-containing compounds during degradation and a gradual shift in the surface properties of the material from hydrophobic to hydrophilic. Simultaneously, the C=O absorption peak of the ester group at 1717 cm⁻¹ significantly weakened, suggesting the breakage of ester bonds in the polyester. Furthermore, the CO stretching vibration peak at 1265 cm⁻¹ decreased synchronously, further confirming the disruption of the polyester backbone structure. In addition, the enhanced C=C vibration peak of the aromatic ring at 1640 cm⁻¹ indicated the formation of aromatic hydrolysis products such as TPA. In summary, the systematic changes in the FT-IR characteristic peaks demonstrate, from a functional group perspective, that strain Jou-S14 can effectively promote the hydrolytic degradation of hcPET-MPs.

[0078] (7) XRD results of degradation of highly crystalline PET microplastics (hcPET-MPs)

[0079] X-ray diffraction (XRD) is a highly efficient method for characterizing changes in the crystalline structure of microplastics and for preliminary identification of degradation effects. By comparing the diffraction peak intensity, peak shape, and the appearance of characteristic diffraction peaks in samples before and after degradation, changes in polymer crystallinity, the disruption of the ordered stacking structure of chain segments, and the formation of degradation products can be clearly revealed. This invention uses XRD analysis to analyze highly crystalline PET microplastics (hcPET-MPs) before and after treatment with the Jou-S14 strain. The results are as follows: Figure 11 As shown, compared with the untreated sample, the intensity of the main diffraction peaks was significantly reduced and the peak shape broadened after treatment, indicating a significant decrease in the crystallinity of hcPET-MPs and a weakening of the ordered crystalline regions. Simultaneously, characteristic diffraction peaks corresponding to degradation products such as BHET, MHET, and TPA were detected in the diffraction pattern, suggesting the generation of corresponding small molecule / monomer products during the degradation process. In summary, the XRD results demonstrate from a crystalline structure perspective that strain Jou-S14 preferentially acts on amorphous regions and further disrupts the ordered stacking structure of polymer chains, thereby promoting the degradation of hcPET-MPs.

[0080] (8) LC-MS results of degradation of highly crystalline PET microplastics (hcPET-MPs)

[0081] Liquid chromatography-mass spectrometry (LC-MS) is a highly efficient method for identifying microplastic degradation products. Qualitative analysis of small-molecule products in the culture supernatant can clearly reveal the formation of intermediates and final products during polyester hydrolysis and subsequent oxidative conversion. This invention uses LC-MS to detect the fermentation broth of hcPET-MPs treated with Jou-S14 strain, and the results are as follows: Figure 12 As shown, various products related to PET degradation were detected in the fermentation broth, including TPA, MHET, phenylacetaldehyde, 2,3-dihydro-1-benzofuran-2-carboxylic acid, diacetylphloroglucinol, and diethylene glycol dibenzoate. These results indicate that strain Jou-S14 can progressively cleave the polyester backbone through hydrolysis, and further transform the degradation products through oxidation, thereby generating aromatic compounds such as MHET and TPA. Simultaneously, the presence of intermediates such as phenylacetaldehyde and diacetylphloroglucinol further confirms that the aromatic ring structure continuously undergoes breakage and transformation during the degradation process. In conclusion, this demonstrates that strain Jou-S14 can effectively degrade hcPET-MPs and possesses significant degradation capabilities.

[0082] The above descriptions are all preferred embodiments of the present invention. For those skilled in the art, any modifications to the present invention in various equivalent forms without departing from the principle of the present invention shall fall within the protection scope of the appended claims.

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 use of Priestia aryabhattai Jou-S14 or its bacterial suspension as described in claim 1 in the degradation of microplastics and their films.

3. The application according to claim 2, 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).

4. A method for degrading polyethylene terephthalate plastic film, characterized in that: The method is as follows: Cut the hcPET film into square segments. First, soak each segment in 2% SDS solution and anhydrous ethanol for more than 4 hours, then rinse 3 times with sterile water. Next, use sterile filter paper to absorb the moisture on the surface of the plastic film on a clean bench and sterilize it with a UV lamp. After sterilization, store the film for 3 days for later use. Then, take 200 μL of the Priestia aryabhattai Jou-S14 bacterial suspension as described in claim 1 and spread it on CFBAM solid medium. Use sterile water as a control group. Place the treated PET plastic film in the center of the petri dish and incubate at 30 ℃ for 30 days.

5. The method according to claim 4, characterized in that: 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 added to CFBAM; LB liquid medium: 10 g tryptone, 10 g sodium chloride, 5 g yeast extract; LB solid medium: 2% agar added to LB liquid medium.

6. 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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