Microplastic degrading bacterium P.ataveovii and application of microplastic degrading bacterium P.ataveovii

Abstract

The invention discloses a micro-plastic degrading bacterium P.ataveovii and application thereof, and belongs to the technical field of microorganisms. According to the invention, a strain of Priestia aryabhattai BK12 is separated and screened from plastic wastes, and the plastic degradation capability of the Priestia aryabhattai BK12 is deeply explored. A research result shows that the P.avenae BK12 can be used for efficiently degrading the PET (Polyethylene Terephthalate), and the P.avenae BK12 can be used for efficiently degrading the PET (Polyethylene Terephthalate), so that the P.avenae BK12 can be used for efficiently degrading the PET (Polyethylene Terephthalate). The invention provides a new microbial resource for the development of a plastic degradation agent, contributes to promoting the development of a biodegradable plastic technology and solving the problem of environmental pollution caused by PET, and assists in realizing the goal of green and sustainable development.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a microplastic-degrading bacterium, *Priscilla argentea*, and its applications. Background Technology

[0002] Polyethylene terephthalate (PET) is a widely used thermoplastic with advantages such as light weight, high transparency, chemical resistance, and recyclability, playing an important role in food packaging, protective films, and product casing manufacturing. However, because plastics are mostly single-use items, the recycling rate is limited, leading to the spread of plastic pollution. Furthermore, PET has extremely high chemical stability and is very difficult to degrade in the natural environment, posing a serious threat to the ecological environment, ecosystem balance, and human health. Given the massive amount of PET waste, traditional landfill and incineration methods are not only inefficient but also cause secondary pollution; therefore, methods for managing plastic waste have always been a major concern. Waste PET products are difficult to degrade in the natural environment, and long-term accumulation can damage soil structure, pollute water bodies, affect the normal growth of crops, and threaten the survival of aquatic life. When PET waste flows into the ocean, it is easily ingested by marine life, leading to their death and disrupting the marine food chain. Furthermore, the microplastics produced from the decomposition of PET can cause long-term harm to biodiversity and may also enter the human body through the food chain, interfering with the endocrine system, damaging the immune system, and posing potential risks to human health. Therefore, it is necessary to provide products that can efficiently degrade PET to reduce plastic pollution and its harm to ecosystems. Summary of the Invention

[0003] The purpose of this invention is to provide a microplastic-degrading bacterium, *Priestia aryabhattai*, and its applications to address the problems existing in the prior art. This invention isolates and screens a strain of *Priestia aryabhattai* BK12 from plastic waste and conducts an in-depth investigation into its ability to degrade plastics. The results show that *Priestia aryabhattai* BK12 can efficiently degrade PET. This invention provides a new microbial resource for the development of plastic degrading agents, contributes to the advancement of biodegradable plastics technology, addresses the environmental pollution caused by PET, and helps achieve the goal of green and sustainable development.

[0004] To achieve the above objectives, the present invention provides the following solution:

[0005] This invention provides a microplastic-degrading bacterium, *Priestia aryabhattai* BK12, which is deposited at the Guangdong Provincial Center for Microbial Culture Collection on May 14, 2025, with accession number GDMCC No:66332.

[0006] The present invention also provides the application of the above-mentioned *Priscilla argentea* BK12 in the preparation of microplastic degrading agents.

[0007] The present invention also provides a microplastic degrading agent comprising the above-mentioned *Priscilla auriculata* BK12 and / or its metabolites.

[0008] The present invention also provides the application of the above-mentioned *Priscilla argentea* BK12 or the above-mentioned microplastic degrading agent in the degradation of plastics.

[0009] Optionally, the plastic includes polyethylene terephthalate.

[0010] The present invention also provides a method for degrading plastics, comprising the step of using the above-mentioned *Priscilla auriculata* BK12 or the above-mentioned microplastic degrading agent to degrade the plastics.

[0011] Optionally, the temperature for degrading the plastic is 28-46 ℃, the pH value is 5-9, and the rotation speed is 150-210 r / min.

[0012] Optionally, the plastic includes polyethylene terephthalate.

[0013] The present invention discloses the following technical effects:

[0014] This invention isolates and screens a strain of *Priestiaaryabhattai* BK12 from plastic waste and conducts an in-depth investigation into its ability to degrade plastics. The results show that *Priestiaaryabhattai* BK12 can efficiently degrade PET at temperatures of 28-46 ℃, pH of 5-9, and rotation speed of 150-210 r / min.

[0015] This invention provides crucial experimental data to support bacterial degradation of plastics, fully demonstrating the feasibility and enormous potential of bacterial degradation of plastics. It provides new microbial resources for the development of plastic degradation agents, contributes to promoting the development of biodegradable plastics technology, solves the environmental pollution problem caused by PET, and helps achieve the goal of green and sustainable development. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 Colony morphology characteristics of strain BK12 on LB solid medium;

[0018] Figure 2 OD200 of strain BK12 for PET degradation at different culture temperatures 600 Lipase activity and weight loss of PET;

[0019] Figure 3 To measure the effect of different incubation speeds on the OD of PET degradation by strain BK12 600 Lipase activity and weight loss of PET;

[0020] Figure 4 OD values ​​of PET degradation by strain BK12 at different pH values 600 Lipase activity and weight loss of PET;

[0021] Figure 5 OD of PET degradation by strain BK12 under optimal culture conditions 600 Lipase activity and weight loss of PET;

[0022] Figure 6 The change in PET degradation rate of strain BK12 over culture time under optimal culture conditions. Detailed Implementation

[0023] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0024] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0025] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0026] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0027] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0028] The technical concept of this invention is as follows:

[0029] Against the backdrop of increasingly severe global plastic pollution, PET, due to its widespread use in packaging, textiles, and other fields, accumulates in the environment in large quantities, posing a significant threat to the ecological environment. This invention focuses on bacterial strains isolated from plastic waste to conduct in-depth research on their degradation effects on PET. To accurately determine the degree of PET degradation, this invention uses PET as the sole carbon source and carefully designs a series of comparative experiments. Under different temperature conditions, covering multiple gradients from low to high temperatures, the degradation ability of the strains on PET is tested, aiming to explore the influence of temperature on the degradation effect. Simultaneously, different incubation speeds are set, gradually varying from low to high, to analyze the correlation between speed and the degree of degradation. Furthermore, studies are conducted under different pH environments, covering multiple pH ranges including acidic, neutral, and alkaline, to comprehensively explore the effect of environmental pH on the degradation of PET by the strains. Through this series of experiments, the optimal environment for the biodegradation of PET by the strains is explored in depth. This not only contributes to a deeper understanding of the mechanism of microbial degradation of PET but also provides important theoretical basis and practical guidance for the development of more advanced polymer biodegradation technologies, and is expected to open up new paths for solving the PET pollution problem and promote technological innovation in the environmental protection field.

[0030] Example 1: Screening and Identification of Strains BK12

[0031] Samples were collected from plastic waste in rivers surrounding Fuyang City and placed in sterilized conical flasks. 100 mL of sterilized deionized water was added, and the flasks were placed in a 37 ℃ constant temperature shaking incubator at 170 rpm for 12 h. The samples were thoroughly shaken and mixed. 5 mL of the extract was pipette-loaded into a sterilized primary screening medium (5.0 g PET powder (crushed to below 300 mesh), 3.0 g glutamic acid, 1.5 g (NH4)2SO4, 1.0 g citric acid, 0.76 g anhydrous K2HPO4, 0.2 g MgSO4, 0.075 g CaCl2, 3.0 g NaCl, 0.015 g MnSO4, 0.022 g ferric ammonium citrate, and 1 L ultrapure water) and incubated at 37 ℃ for four weeks. 200 μL of the culture medium from the initial screening medium was transferred to LB solid medium, and the culture was repeatedly isolated and purified using the streak plate method to obtain single colonies. The lipase activity of the single colony strain was measured to preliminarily assess the strain's degradation effect on PET.

[0032] The method for determining lipase activity (p-nitrophenol method) is as follows:

[0033] Lipase hydrolyzes p-nitrophenol esters to produce p-nitrophenol (p-NP). Under alkaline conditions, p-NP is yellow and has an absorbance at 405 nm. Lipase activity can be assessed by measuring the absorbance. A p-nitrophenol ester (such as p-nitrophenol palmitate) was dissolved in 50 mM Tris-HCl (pH 8.0) buffer to prepare the substrate solution. A 3000 μL reaction system was prepared: 2.7 mL buffer, 200 μL p-nitrophenol ester substrate, and 100 μL of the test bacterial culture. The substrate and buffer were preheated at 37°C for 2 min, then the test bacterial culture was added and reacted for 5 min. Finally, 100 μL of 0.1 mmol / L Na₂CO₃ was added to terminate the reaction, and the OD value was measured at 405 nm. Distilled water was used as a blank control, and an inactivated test bacterial culture (with other components identical to the system) was also used as a control, with its OD value measured. Changes in OD values ​​reflect lipase activity.

[0034] Finally, a strain BK12 capable of producing high levels of lipase was selected. On LB agar, the colonies of this strain were light-colored, almost milky white or light beige; they were round or nearly round, with relatively clear boundaries and regular morphology; the surface was smooth, and some colonies showed slightly more pronounced elevation, exhibiting a plump appearance. Figure 1 ).

[0035] Strain BK12 was sent to Beijing Ruibo Biotechnology Co., Ltd. for sequencing. The 16S rDNA partial sequence homology of the strain was compared using BLASTN software and the EZbiocloud website. A phylogenetic tree was constructed using MEGA 5.0. Strain BK12 was identified as *Priestia aryabhattai*, and its 16S rDNA sequence is as follows:

[0036]

[0037] The strain Priestia aryabhattai BK12 was deposited on May 14, 2025, at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCC No:66332.

[0038] Example 2: Optimization of conditions for PET degradation by strain BK12

[0039] 1. Temperature

[0040] To investigate the effect of ambient temperature on the degradation of PET by strain BK12, the shaking incubator temperatures were set at 28 ℃, 37 ℃, 46 ℃, 55 ℃, and 60 ℃. BK12 cells were inoculated into 100 mL of primary screening medium with PET as the sole carbon source, and then cultured on a shaker at 170 r / min for 4 days. After culture, the OD was measured. 600 The activity of lipase and the weight loss of PET were measured to evaluate the degradation effect of microbial strains on PET under different temperature conditions.

[0041] The results are as follows Figure 2 As shown, temperature significantly affects the PET degradation ability of strain BK12; both excessively high and low temperatures inhibit its degradation activity. When the temperature is below 37 ℃ or above 46 ℃, the PET degradation rate decreases significantly. This may be because low temperatures reduce enzyme activity and slow microbial metabolism, thus affecting degradation efficiency. High temperatures (>46 ℃) may cause denaturation and inactivation of key degrading enzymes (such as PET lipase), and even damage cell growth, thereby weakening the degradation ability. Within the temperature range of 28-46 ℃, the strain exhibits strong PET degradation ability, indicating that this temperature range is suitable for it to exert its optimal degradation activity. Further analysis revealed that 37 ℃ is the optimal degradation temperature for the strain, at which the PET degradation rate of BK12 reaches 22.19±2.12%. This result is consistent with the optimal temperature pattern for microbial growth and enzyme activity. 600 The value was 1.73±0.08, and the lipase activity reached 59.53±3.26 U / mL. In summary, in practical applications, to maximize the PET degradation efficiency of the strain, the culture temperature should be controlled between 28-46 ℃, with 37 ℃ being the preferred temperature for optimal degradation.

[0042] 2. Rotation speed

[0043] To investigate the effect of culture rotation speed on the degradation of PET by strain BK12, five different culture rotation speed gradients were set up in this experiment: 130 r / min, 150 r / min, 170 r / min, 190 r / min, and 210 r / min. BK12 cells were inoculated into 100 mL of primary screening medium with PET as the sole carbon source. The inoculated medium was then placed in a constant-temperature shaker and cultured for 4 consecutive days at 37 ℃ and rotation speeds of 130 r / min, 150 r / min, 170 r / min, 190 r / min, and 210 r / min. After the culture period, OD was measured. 600 The activity of lipase and the weight loss of PET were measured to accurately assess the degradation ability of microbial strains with different inoculation speed gradients on PET.

[0044] The results are as follows Figure 3 As shown, the rotation speed significantly affects the degradation efficiency of the strain. The degradation rate is highest at a rotation speed of 170 r / min, with a PET degradation rate of 19.54 ± 1.70%. Within the rotation speed range of 150–210 r / min, the strain exhibits strong PET degradation ability, indicating that this rotation speed range is suitable for its optimal degradation activity. This result is consistent with the optimal temperature pattern for microbial growth and enzyme activity. 600 The value was 1.88±0.09, and the lipase activity reached 49.03±5.75 U / mL. In summary, in practical applications, to maximize the PET degradation efficiency of the strain, the culture speed should be controlled between 150-210 r / min, with 170 r / min being the preferred speed for achieving the best degradation effect.

[0045] 3. Initial pH

[0046] To accurately investigate the effect of initial pH on the degradation of PET by strain BK12, five different initial pH gradients were set up: 3.0, 5.0, 7.0, 9.0, and 11.0. Under each pH condition, BK12 cells were inoculated into 100 mL of primary screening medium with PET as the sole carbon source. The inoculated medium was then placed in a shaker at a constant temperature of 37 ℃ and a rotation speed of 170 r / min for continuous shaking incubation for 4 days. After incubation, OD was measured. 600 The activity of lipase and the weight loss of PET were studied to scientifically evaluate the degradation effect of microbial strains on PET under different initial pH values.

[0047] The results are as follows Figure 4As shown, the degradation ability of the strain first increases and then decreases with increasing initial pH value, indicating that the strain's ability to degrade PET is less affected within the weakly acidic and weakly alkaline range, i.e., pH 5-9. At pH=7, OD... 600 It has the highest value and the greatest degradation ability, with a PET degradation rate of 18.42±2.20% and OD. 600 The pH was 1.85 ± 0.09, and the lipase activity reached 67.97 ± 4.52 U / mL. Within the pH range of 5-9, the strain exhibited strong PET degradation ability, indicating that this culture pH range is suitable for its optimal degradation activity. This result is consistent with the optimal temperature pattern for microbial growth and enzyme activity, suggesting that pH 5-9 may be the optimal reaction pH for the strain's lipase. In summary, in practical applications, to maximize the PET degradation efficiency of the strain, the culture pH should be controlled between 5-9, with pH=7 being the preferred choice for achieving the best degradation effect.

[0048] Example 3: Determination of PET degradation ability of strain BK12

[0049] The optimal pH, reaction temperature, and culture speed selected in Example 2 were used as the culture conditions for the degradation of PET by strain BK12. BK12 cells were inoculated into 100 mL of initial screening medium with PET as the sole carbon source. The inoculated medium was then placed in a shaker with an initial pH of 7, a constant temperature of 37 °C, and a rotation speed maintained at 170 r / min for 15 days to investigate the effect of the microbial strain on PET degradation. On day 15, 40 mL of a 2×10⁻⁶ solution was added to the culture medium. 8 CFU / mL bacterial culture.

[0050] The results are as follows Figure 5 and Figure 6 As shown, after 15 days of culture, strain BK12 achieved a PET degradation rate of 30.25±1.39%. With a second addition of bacterial culture, the degradation rate reached 52.96±1.76% after 30 days.

[0051] In this invention, *Priestiaaryabhattai* BK12 was successfully isolated and screened from plastic waste, and its ability to degrade PET was investigated in depth. The results showed that temperature, pH, and incubation speed had a significant impact on the strain's ability to degrade PET. The optimal degradation temperature was 37 °C, a neutral environment (pH = 7) was most favorable for the degradation reaction, and the strain's degradation ability reached its peak at an incubation speed of 170 r / min.

[0052] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A microplastic-degrading bacterium, *Priestia aryabhattai* BK12, characterized in that, It is deposited at the Guangdong Provincial Center for Microbial Culture Collection on May 14, 2025, with accession number GDMCC No:66332.

2. The use of *Priscilla argentea* BK12 as described in claim 1 in the preparation of microplastic degrading agents.

3. A microplastic degrading agent, characterized in that, The microplastic degrading agent comprises *Priscilla auriculata* BK12 as described in claim 1 and / or its metabolites.

4. The application of *Priscilla auriculata* BK12 as described in claim 1 or the microplastic degrading agent as described in claim 3 in the degradation of plastics.

5. The application according to claim 4, characterized in that, The plastic includes polyethylene terephthalate.

6. A method for degrading plastics, characterized in that, The step includes using the *Priscilla auriculata* BK12 as described in claim 1 or the microplastic degrading agent as described in claim 3 to degrade plastics.

7. The method according to claim 6, characterized in that, The temperature for degrading the plastic is 28-46 ℃, the pH value is 5-9, and the rotation speed is 150-210 r / min.

8. The method according to claim 6, characterized in that, The plastic includes polyethylene terephthalate.

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