Enterobacter sp. capable of degrading polypropylene and application thereof

Abstract

The application discloses a soil enterobacter capable of efficiently degrading polypropylene and application thereof. The strain is FJNUIM-S2, which is classified and named as soil enterobacter (Enterobacter agglomerans) Enterobacter soli ), is preserved in the China General Microbiological Culture Collection Center (CGMCC), and is preserved on October 17, 2024, with a preservation number of CGMCC No. 32241 and a preservation address of No. 3, Institute of Microbiology, Chinese Academy of Sciences, Xiliujiaobei, Chaoyang District, Beijing. The strain FJNUIM-S2 of the application can grow with polypropylene as the only carbon source, has good polypropylene degradation characteristics, provides a microbial resource for bioremediation of waste polypropylene in the environment, and has a wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a highly efficient soil enterobacter strain that degrades polypropylene and its applications. Background Technology

[0002] Plastic products are widely used in various fields due to their low production cost and durability. With the development of the plastics industry and the widespread use and consumption of plastic products, an increasing number of waste petroleum-based plastic products are entering the environment. Conventional physical and chemical methods for treating plastic waste have limitations such as high cost, numerous byproducts, and secondary pollution, making "white pollution" a global problem. Polyethylene (PE), polypropylene (PP), and polystyrene (PS) plastics account for 50% of the total consumption of synthetic plastics and are the main components of plastic waste. Polypropylene is widely used in masks, packaging, and packaging boxes, accounting for 18.9% of global plastic production in 2022, making it the second largest plastic after polyethylene. Furthermore, polypropylene is chemically inert than polyethylene, making it more difficult to degrade.

[0003] The recycling technology for waste polypropylene is limited, resulting in low added value of recycled products and failing to realize the value of reuse. Waste plastics in the environment cause severe damage to ecosystems; the formation of microplastics and nanoplastics under abiotic or biotic conditions further endangers the health of plants, animals, and humans. Incineration produces harmful gases, while landfill disposal has a long degradation cycle and seriously impacts soil ecology. Currently, there is an urgent need for environmentally friendly, efficient, and simple degradation methods to alleviate the global pollution problem caused by polypropylene plastics, making microbial degradation of plastics a focus of attention.

[0004] Among the reported microorganisms capable of degrading plastics, those targeting polyethylene (PE) and polyethylene terephthalate (PET) are mainly bacteria of the genera *Pseudomonas* and *Bacillus*, and fungi of the genera *Aspergillus* and *Fusarium*. Microorganisms with high plastic degradation capabilities include *Pseudomonas* strains screened by Balasubramanian et al. that can efficiently degrade high-density polyethylene (HDPE). Pseudomonas sp. GMB7 and Arthrobacterium Arthrobactersp. GMB5, after 30 days of incubation, lost 15% and 12% of their weight, respectively (Balasubramanian V, Natarajan K, Hemambika B, et al. High-density polyethylene (HDPE)-degrading potential bacteria from marineecosystem of Gulf of Mannar, India. Lett Appl Microbiol 2010, 51(2): 205-211.); Elsamahy et al. constructed a strain isolated from termite gut. Steri gmatomyces halophilus SSA1575 Meyerozyma guilliermondii SSA1547 and Meyerozyma caribbica A yeast consortium composed of SSA1654 caused a 63.4% reduction in tensile strength and a 33.2% reduction in net low-density polyethylene (LDPE) mass (Elsamahy T, Sun J, Elsilk SE, et al. Biodegradation of low-density polyethylene plastic waste by a constructed tri-culture yeast consortium from wood-feeding termite: Degradation mechanism and pathway. Hazard Mater 2023, 448: 130944.); Auta et al. discovered Bacillus... Bacillus cereus and Bacillus gottheilii After 40 days of shake-flask experiments, the weight loss of PET was 6.6% and 3.0%, respectively (Auta HS, Emenike CU, Fauziah SH. Screening of Bacillus (Strains isolated from mangrove ecosystems in Peninsular Malaysia for microplastic degradation. Environ Pollut 2017, 231(Pt2): 1552-1559.) Das et al. screened out two bacterial strains. Pseudomonas aeruginosa NBTU01 and Achromobactersp. NBTU02 showed degradation rates of 35.65% and 34.63% for PVC after 180 days (Das G, Bordoloi NK, Rai SK, et al. Biodegradable and biocompatible epoxidized vegetable oil modified thermostable poly(vinyl chloride): thermal and performance characteristics post biodegradation with Pseudomonas aeruginosa and Achromobacter sp. Hazard Mater 2012, 209-210: 434-442.); Ganesh et al. screened a strain of Bacillus. Bacillus paralicheniformis G1 degraded 34% of the PS membrane within 60 days (Ganesh Kumar A, Hinduja M, Sujitha K, et al. Biodegradation of polystyrene by deep-sea Bacillus paralicheniformis G1 and genome analysis. Science Total Environ 2021, 774: 145002.); Jeon et al. screened bacteria Stenotrophomonas panacihumi After 90 days of composting, PA3-2 showed a degradation rate of 20.3±1.39% for LMWPP-1 (Mn: 2,800, Mw: 10,300) and 16.6±1.70% for LMWPP2 (Mn: 3,600, Mw: 19,700) (Jeon HJ, Kim MN. Isolation of mesophilicbacterium for biodegradation of polypropylene. International Biodeterioration & Biodegradation 2016, 115: 244-249.). Skariyachan et al. isolated 8 strains of plastic-degrading bacteria, among which... Aneurinibacillus aneurinilyticus btDSCE01、 Brevibacillus agri btDSCE02 Brevibacillus sp. btDSCE03 and Brevibacillus brevisThe btDSCE04 consortium exhibited the highest degradation rate, exceeding that of individual consortia. After 140 days of cultivation at 50°C, the degradation rate of PP particles reached 56.3 ± 2% (Skariyachan S, Patil AA, Shankar A, et al. Enhanced polymer degradation of polyethylene and polypropylene by novel thermophilic consortia of Brevibacillus sps. and Aneurinibacillus sp. screened from waste management landfills and sewage treatment plants. Polymer Degradation and Stability 2018, 149: 52-68.). These degrading strains demonstrate the promising future of biodegradable plastics and lay the foundation for the discovery of possible degradation mechanisms of highly efficient degrading strains and highly efficient degrading enzymes.

[0005] However, there are very few reports on the biodegradation of polypropylene plastics, and there is a severe lack of polypropylene plastic degrading strains that can be studied and applied. Therefore, finding effective microbial and enzyme resources for biodegrading polypropylene plastics, enriching the resource library of degrading strains, and developing and improving green recycling processes for biodegrading polypropylene are of great significance to the environmental pollution caused by polypropylene plastics. Summary of the Invention

[0006] The purpose of this invention is to develop a highly efficient soil enterobacter strain that degrades polypropylene and its applications.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] This invention first provides a strain of polypropylene that is highly efficient at degrading polypropylene, FJNUIM-S2, which is classified and named Enterobacter stolonifera (…). Enterobacter soli The sample was deposited at the China General Microbiological Culture Collection Center (CGMCC) on October 17, 2024, with accession number CGMCC No. 32241. The deposit address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0009] The nucleotide sequence of the 16S rDNA of strain FJNUIM-S2 is shown in SEQ ID No. 1.

[0010] The strain FJNUIM-S2 can be used to biodegrade polypropylene, especially polypropylene plastics, including polypropylene plastic films, granules or powders.

[0011] The present invention also provides the application of the strain FJNUIM-S2 in the preparation of a degrading agent for polypropylene degradation.

[0012] This invention also provides a polypropylene-degrading bacterial agent produced by strain FJNUIM-S2, the preparation method of which includes the following steps:

[0013] (1) After streaking and activating the strain FJNUIM-S2 on an LB plate, pick single clones and inoculate them into a test tube containing LB liquid medium. Culture them until the exponential growth phase to obtain the primary seed culture. Then, inoculate the strain into LB liquid medium at an inoculation amount of not less than 0.5% (v / v) and culture with shaking until the exponential growth phase to obtain the seed culture for fermentation.

[0014] (2) Inoculate the seed culture for fermentation into the culture medium of the seed tank at an inoculation amount of 5-20% (v / v) and culture until the exponential growth phase to obtain the secondary seed culture;

[0015] (3) Inoculate the secondary seed culture at an inoculation rate of not less than 0.2% (v / v) into the culture medium of the production tank and ferment for 8-12 hours. During the fermentation process, adjust the pH online to around 7.0, maintain a sterile air flow rate of 1-2 vvm, a stirring rate of 200-400 rpm, and a culture temperature of 30-37℃. After fermentation, the culture medium is directly dispensed into liquid degradation agent, or the bacterial cells are dried into powder, or a high-purity preparation is prepared through separation and purification steps. The culture medium in the seed tank is LB medium, and the culture medium in the production tank is a polypropylene powder (membrane) single carbon source medium.

[0016] The polypropylene degradation agent can be used to biodegrade polypropylene plastics, wherein the polypropylene plastics are polypropylene plastic waste existing in landfills or water bodies.

[0017] The present invention also provides a method for polypropylene degradation, the method comprising culturing the strain FJNUIM-S2 or the polypropylene degradation agent, and contacting the polypropylene plastic to be degraded with the strain culture or the degradation agent.

[0018] Compared with existing technologies, the polypropylene-efficient degrading strain FJNUIM-S2 obtained by the present invention can be used for the microbial degradation of polypropylene, especially waste polypropylene films, granules, and powdered polypropylene plastics in the environment. The strain FJNUIM-S2 of the present invention can grow using polypropylene as the sole carbon source, exhibits excellent polypropylene degradation characteristics, and provides microbial resources for the bioremediation of waste polypropylene in the environment, showing broad application prospects.

[0019] The strain FJNUIM-S2 of this invention exhibits excellent polypropylene degradation ability, especially for polypropylene films. When using this strain to degrade polypropylene plastics, after 60 days of treatment, the surface of the polypropylene film becomes noticeably rough, with obvious pores, grooves, and depressions, resulting in severe surface damage. The weight loss of the degraded polypropylene film before and after degradation can reach approximately 0.5 mg. Furthermore, the thermal decomposition temperature of the degraded polypropylene film decreases, and its thermal stability deteriorates. This degradation method is environmentally friendly, has low degradation cost, and is simple and convenient to operate.

[0020] The degradation agent of the present invention can be produced using fermentation equipment commonly used in the fermentation industry. It has the advantages of good degradation effect, low production cost and convenient operation. It is suitable for treating waste polypropylene that is difficult to degrade in the environment and has positive significance in protecting the ecological environment and maintaining human health. Attached Figure Description

[0021] Figure 1 This is the morphology of a single colony of strain FJNUIM-S2 after plate dilution.

[0022] Figure 2 This is a phylogenetic tree constructed based on the 16S rRNA sequence, in which strain FJNUIM-S2 is used. Annotation.

[0023] Figure 3 This is the growth curve of *Enterobacter spp.* FJNUIM-S2, with polypropylene as the sole carbon source.

[0024] Figure 4 These are the surface microstructures of polypropylene membranes before and after degradation under a scanning electron microscope. (A) Control group (no inoculation); (B) Polypropylene membrane degraded by Enterobacter spp. FJNUIM-S2.

[0025] Figure 5 This is a Fourier transform infrared spectroscopy analysis of the polypropylene film before and after degradation.

[0026] Figure 6 It is a thermogravimetric analysis of polypropylene film before and after degradation. Detailed Implementation

[0027] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0028] Unless otherwise specified, all raw materials and reagents used in this invention are commercially available.

[0029] Example 1: Isolation, screening and identification of strain FJNUIM-S2

[0030] 1. Isolation and screening of polypropylene-degrading bacteria

[0031] 1.1 Sample collection: Soil samples were collected from the Wengcuoshan landfill (old site) in Minhou County, Fujian Province. The collection depth was 3-10 cm. The collected soil samples were placed in sterile Erlenmeyer flasks and brought back to the laboratory for later use.

[0032] 1.2 Experimental culture medium

[0033] (1) LB medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, and 15-20 g / L agar powder for solid medium. Add double-distilled water to 1000 mL, adjust pH to 7.2 with 5 mol / L NaOH, and sterilize at 121℃ for 30 min.

[0034] (2) 10× Trace element stock solution: Weigh 10 g FeSO4·7H2O, 10 g MgSO4·7H2O, 1 g MnSO4·H2O, 1 g ZnSO4·7H2O, and 1 g CuSO4·5H2O into 800 mL of double-distilled water, stir until completely dissolved, and then bring the volume to 1000 mL. Filter through a 0.22 μm filter membrane to remove bacteria.

[0035] (3) Microcarbon source culture medium: Weigh 0.5 g yeast extract, 2 g (NH4)2SO4 and 4.5 g polypropylene powder into 800 mL double-distilled water, stir until completely dissolved, and then bring the volume to 900 mL. Sterilize at 121 °C for 15 min. After cooling, add 100 mL of 10× micronutrient stock solution.

[0036] (4) Polypropylene powder as the sole carbon source culture medium: Weigh 2 g (NH4)2SO4 and 5 g polypropylene powder into 800 mL of double-distilled water, stir until completely dissolved, and then bring the volume to 900 mL. Sterilize at 121℃ for 15 min. After cooling, add 100 mL of 10× trace element stock solution.

[0037] (5) Polypropylene membrane as the sole carbon source medium: Weigh 0.1 g (NH4)2SO4 into 40 mL of double-distilled water, stir until completely dissolved, and then bring the volume to 45 mL. Sterilize at 121 °C for 15 min. After cooling, add the pretreated polypropylene membrane and 5 mL of 10× trace element stock solution.

[0038] 1.3 Isolation of strains

[0039] Add 2 g of collected soil sample to 50 mL of micro-carbon source medium. Use a micro-carbon source medium without inoculation as a blank control, incubating under the same conditions at 37℃ and 180 rpm for 6 days. Collect bacterial cells by centrifugation at 4℃ and 5000 rpm. Transfer the collected bacterial pellet to a fresh 50 mL micro-carbon source medium and continue incubation for 6 days. Collect bacterial cells by centrifugation at 4℃ and 5000 rpm, and transfer to 50 mL of polypropylene powder as the sole carbon source medium. Use a polypropylene powder as the sole carbon source medium without inoculation as a blank control, incubating at 37℃ and 180 rpm for 10 days. Collect bacterial cells by centrifugation at 4℃ and 5000 rpm, resuspend the cells in 200 μL of physiological saline, and spread on LB agar plates. Incubate at 37℃ for 12-24 h. After colony growth, pick colonies with different morphologies and inoculate them onto LB agar plates. Repeat streaking to separate until single colonies are obtained. After single colonies have grown, they are transferred to new LB slant agar plates for preservation to obtain the purified strain FJNUIM-S2. Meanwhile, the bacterial culture in liquid form is preserved in LB agar containing 30% glycerol at -80°C for later use.

[0040] 1.4 DNA extraction and PCR amplification of the strain

[0041] Collect 5 mL of bacterial culture incubated overnight at 37°C and extract DNA according to the procedure of the bacterial genomic DNA extraction kit (Nanjing Novizan Biotechnology Co., Ltd., catalog number: DC103-01). Store the extracted bacterial DNA at -20°C for later use. Using the extracted genome as a template, amplify bacterial 16S rRNA using universal primers 27F and 1492R (primers 27F: 5'-AGAGTTTGATCCTGGCTCAG-3' and 1492R: 5'-TACGGCTACCTTGTTACGACTT-3' were synthesized by Beijing Qingke Biotechnology Co., Ltd.). The PCR amplification reaction system was 20 μL: 1 μL DNA template, 0.8 μL each of primers 27F and 1492R (10 μM), 10 μL of 2×Phanta Max MasterMix (Nanjing Novizan Biotechnology Co., Ltd., catalog number: P515-01), and ddH2O to a total volume of 20 μL. The PCR reaction conditions were as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 56℃ annealing for 30 s, 72℃ extension for 1 min, for a total of 30 cycles; 72℃ final extension for 5 min, and storage at 4℃. The qualified PCR products were sent to Fuzhou Qingke Biotechnology Co., Ltd. for sequencing.

[0042] 2. Identification of strains

[0043] After the purified strain FJNUIM-S2 was inoculated onto LB solid medium and cultured at 37°C for 1 day (3 replicates), the colony morphology was described, and the microscopic morphology was measured and described after slide preparation, as follows:

[0044] Morphological description of strain FJNUIM-S2: The morphology of this bacterium was observed and recorded after culturing on LB solid medium for 1 day. The colonies were round, 1-2 mm in size, white and opaque on the front, with neat edges, a raised center, a smooth surface, and a moist texture. Figure 1 ).

[0045] The purified strain FJNUIM-S2 was inoculated into LB liquid medium and cultured at 37°C and 180 rpm until the exponential growth phase. Gram staining was performed, and the strain was observed to be Gram-negative under an optical microscope, showing that it stained red.

[0046] Based on the 16S rRNA sequence, a sequence with high similarity to strain FJNUIM-S2 was downloaded. ClustalX2 software was used to align the strain with the downloaded sequence, and MEGA11 software was used to perform alignment with Bacillus subtilis. Bacillus sp. Strain A6 was an outgroup. A phylogenetic tree was constructed between strain FJNUIM-S2 and related strains using the maximum likelihood method. The results showed that the sequence of this strain clustered well with the sequences of Enterobacteriaceae. Figure 2 The BLASTn alignment results using 16S rRNA sequence showed that strain FJNUIM-S2 was similar to... Enterobacter soli The similarity was as high as 99.93%. Based on the combined morphological characteristics and molecular biological experimental data, strain FJNUIM-S2 was identified as belonging to *Enterobacter tumefaciens*. Enterobacter soli .

[0047] The soil Enterobacter FJNUIM-S2 of the present invention ( Enterobacter soli FJNUIM-S2 is deposited at the China General Microbiological Culture Collection Center (CGMCC) on October 17, 2024, with accession number CGMCC No. 32241. The deposit address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0048] 3. Preservation and subculturing of strains:

[0049] The *Enterobacter spp.* FJNUIM-S2 strain was activated by streaking on LB agar plates and incubated upside down at 37°C for one day. Single colonies were then picked and inoculated into LB liquid medium and incubated overnight at 37°C and 180 rpm. Under aseptic conditions, the cultures were transferred to sterilized 30% glycerol cryovials. Three vials were stored at -20°C and three vials were stored at -80°C. The survival of the strains was checked periodically.

[0050] To evaluate the degradation ability of polypropylene-degrading bacteria on waste polypropylene plastics in the environment, polypropylene powder was selected as the sole carbon and energy source in the culture medium, and the growth curves of the degrading microorganisms were determined.

[0051] Enterobacter spores FJNUIM-S2 were inoculated at a rate of 2% into a polypropylene powder-based medium with polypropylene powder as the sole carbon source and mixed thoroughly. A control group without inoculated bacteria was used as the sole carbon source. The initial OD was measured using the control group as a blank for zeroing. 600 Value. Cultured at 37℃ and 180 rpm, with OD values ​​measured at fixed points every 3 days. 600 Growth curves of the strain were plotted on polypropylene as the sole carbon source medium. The growth curves show that *Enterobacter stolonifer* FJNUIM-S2, when grown on polypropylene powder as the sole carbon source medium, was in a lag phase for the first 9 days, entered the exponential growth phase on day 9, and remained there until the stationary phase on day 18. Figure 3 ).

[0052] 1.1 Determination of weight loss of polypropylene film

[0053] Polypropylene membranes with a thickness of 0.3 mm were cut into 1.5 × 1.5 cm pieces, soaked in 2% SDS for 4 hours, ultrasonically cleaned for 30 minutes, and then transferred to a clean bench. After soaking in 75% alcohol for 4 hours, they were rinsed three times with sterile double-distilled water and air-dried in the clean bench. The initial weight was measured using a 0.01% balance and recorded. The membranes were then labeled and numbered, and the corresponding polypropylene membrane-only carbon source medium was prepared. *Enterobacter stolonifer* FJNUIM-S2 was inoculated into the polypropylene membrane-only carbon source medium at a 2% inoculum rate. Each strain was divided into three replicates, with the polypropylene membrane-only carbon source medium without inoculation serving as a control. The membranes were cultured in shake flasks at 37°C and 100 rpm for 60 days. After the shake flask culture, the polypropylene membranes were removed, and the remaining weight of the degraded polypropylene membrane was measured using a 0.01% balance. The weight loss of the polypropylene membrane after degradation by *Enterobacter stolonifer* FJNUIM-S2 strain was approximately 0.5 mg.

[0054] 1.2 Scanning electron microscopy observation of the surface of the polypropylene film

[0055] The degraded polypropylene membrane was cut into 2 mm × 2 mm pieces at the center, treated with gold sputtering twice, and then observed. The sample was located under low magnification, then switched to high magnification mode, and the brightness and contrast were manually adjusted until the sample morphology was clear before slow scanning. After determining the desired sample morphology, the image was taken and saved. After the degraded polypropylene membrane was treated with gold sputtering twice, it was observed. Compared with the control group (uninoculated), the surface of the polypropylene membrane degraded by *Enterobacter spp.* FJNUIM-S2 was significantly rougher, with obvious pores, grooves, and depressions, indicating greater damage. Figure 4 ).

[0056] 1.3 Fourier Transform Infrared Spectroscopy for Detection of Polypropylene Membrane

[0057] The degraded polypropylene film was cut into small fragments and labeled for later use. The sample carrier, spoon, and abrasive were wiped with alcohol swabs and dried in an oven. The sample to be tested and potassium bromide powder were ground in the abrasive at a ratio of 1:50. The ground mixture was placed into the sample carrier, then placed on a tablet press and slightly tightened to secure it. After adjusting the pressure to 1.5, it was allowed to rest briefly before loosening. The sample carrier was then removed and placed in the center of the infrared chamber. Fourier transform infrared spectroscopy was then used to collect sample data. The control group consisted of PP (untreated polypropylene film) and PP / shaking (uninoculated polypropylene film). The PP / shaking sample was at ~1635 cm⁻¹. -1 and ~3448 cm -1 The presence of a distinct absorption peak is likely due to trace elements in the culture medium being firmly adsorbed onto the polypropylene during the shake-flask process and unable to be washed off. The polypropylene membrane degraded by *Enterobacter spp.* FJNUIM-S2 showed an absorption peak at ~3435 cm⁻¹. -1 The characteristic peak absorption intensity of the hydroxyl group (-OH) at this position is relatively high. Figure 5 It is speculated that the polypropylene membrane degraded by *Enterobacter spp.* FJNUIM-S2 may have undergone a hydroxylation reaction.

[0058] 1.4 Thermogravimetric analysis of polypropylene film

[0059] The degraded polypropylene membrane was cut into 2 mm × 2 mm pieces, and 5-10 mg was weighed and dried at 85℃ for 4 hours. After loading the sample, the crucible was placed in a tray, ensuring the tray was stable, and then thermogravimetric analysis was started. The thermogravimetric curve of the polypropylene membrane degraded by *Enterobacter sericultures* FJNUIM-S2 showed a significant leftward shift compared to the control group (PP: untreated polypropylene membrane; PP / shaking: uninoculated polypropylene membrane). Figure 6 The results indicate that the thermal decomposition temperature of the degraded polypropylene film decreases, and its thermal stability deteriorates; this also shows that *Enterobacter spp.* FJNUIM-S2 can destroy the molecular structure of the polypropylene film, thus reducing its thermal stability.

[0060] (1) After streaking and activating the strain FJNUIM-S2 on an LB solid medium plate, pick single clones and inoculate them into a test tube containing LB liquid medium. Culture them until the exponential growth phase to obtain the primary seed culture. Then, inoculate the strain into LB liquid medium at an inoculation amount of not less than 0.5% (v / v) and culture with shaking until the exponential growth phase to obtain the seed culture for fermentation.

[0061] (2) Inoculate the seed culture for fermentation into the culture medium of the seed tank at an inoculation amount of 5-20% (v / v) and culture until the exponential growth phase to obtain the secondary seed culture;

[0062] (3) Inoculate the secondary seed culture at an inoculation rate of not less than 0.2% (v / v) into the culture medium of the production tank and ferment for 8-12 hours. During the fermentation process, adjust the pH online to around 7.0, maintain a sterile air flow rate of 1-2 vvm, a stirring rate of 200-400 rpm, and a culture temperature of 30-37℃. After fermentation, the culture medium is directly dispensed into liquid degradation agent, or the bacterial cells are dried into powder, or a high-purity preparation is prepared through separation and purification steps. The culture medium in the seed tank is LB medium, and the culture medium in the production tank is a polypropylene powder (membrane) single carbon source medium.

[0063] The microbial agent prepared in Example 4 of this invention can react directly with polypropylene plastic films, particles, or powders, and can also effectively degrade polypropylene plastics to achieve the effects of Examples 2 and 3.

[0064] The results in summary indicate that *Enterobacter stolonifer* FJNUIM-S2 has a good effect on degrading polypropylene plastics without pretreatment.

[0065] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any simple modifications, equivalent changes, and alterations made to the above embodiments without departing from the technical essence of the present invention shall still fall within the scope of the present invention.

Claims

1. A strain of polypropylene that is highly efficient at degrading polypropylene, FJNUIM-S2, characterized in that, Its classification name is Enterobacter stropharia ( Enterobacter soli The sample was deposited at the China General Microbiological Culture Collection Center (CGMCC) on October 17, 2024, with accession number CGMCC No. 32241. The deposit address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

2. The application of strain FJNUIM-S2 as described in claim 1 in the biodegradation of polypropylene.

3. The application according to claim 2, characterized in that, The polypropylene mentioned includes polypropylene plastic films, granules, or powder.

4. The application of strain FJNUIM-S2 as described in claim 1 in the preparation of a degrading agent for polypropylene.

5. A polypropylene-degrading bacterial agent produced using the strain FJNUIM-S2 described in claim 1.

6. The method for preparing the polypropylene degrading microbial agent as described in claim 5, characterized in that, Includes the following steps: (1) After the strain FJNUIM-S2 described in claim 1 is isolated and activated by streaking on an LB plate, a single clone is picked and inoculated into a test tube containing LB liquid medium and cultured until the exponential growth phase to obtain the primary seed liquid; then, the inoculation amount is not less than 0.5% (v / v) into LB liquid medium and cultured with shaking until the exponential growth phase to obtain the seed liquid for fermentation. (2) Inoculate the seed culture for fermentation into the culture medium of the seed tank at an inoculation amount of 5-20% (v / v) and culture until the exponential growth phase to obtain the secondary seed culture; (3) Inoculate the secondary seed liquid into the culture medium of the production tank at an inoculation amount of not less than 0.2% (v / v) and ferment for 8-12 hours. During the fermentation process, adjust the pH to about 7.0 online, the ventilation rate of sterile air is 1-2 vvm, the stirring rate is 200-400 rpm, and the culture temperature is 30-37℃. After the fermentation is completed, the culture liquid is discharged from the tank and packaged to obtain liquid degradation bacterial agent.

7. The method for preparing the polypropylene degrading microbial agent according to claim 6, characterized in that, The culture medium after fermentation in step (3) is dried to make powder, or it is prepared into a high-purity preparation through separation and purification.

8. The application of the polypropylene degrading microbial agent as described in claim 5 in the biodegradation of polypropylene plastics.

9. The application according to claim 8, characterized in that, The polypropylene plastic mentioned is polypropylene plastic waste that exists in landfills or water bodies.

10. A method for the degradation of polypropylene, characterized in that, The method includes culturing strain FJNUIM-S2 as described in claim 1 or polypropylene degrading agent as described in claim 5, and contacting the polypropylene plastic to be degraded with the strain culture or degrading agent.

Images