PET pretreatment method and application of PET pretreatment method in promotion of bacterial degradation

Abstract

The invention discloses a PET (Polyethylene Terephthalate) pretreatment method and application thereof in promoting bacterial degradation. The mesorhizobium sp. SCSIO 43976 is preserved in the Guangdong Microbial Culture Collection Center on October 24, 2025, and the preservation number of the mesorhizobium sp. SCSIO 43976 is GDMCC No: 67156. Sorhizobium sp. SCSIO 43976 in bacteria which are derived from a marine ecosystem and have high-efficiency PET degradation capability, a set of high-temperature melting-low-temperature cold quenching pretreatment method which is matched with the Sorhizobium sp. SCSIO 43976 and can effectively reduce the crystallization degree of PET, and a synergistic degradation system formed by the Sorhizobium sp. SCSIO 43976 and the Sorhizobium sp. SCSIO 43976 are used for finally realizing the purpose of degrading the waste PET by using the Sorhizobium sp. SCSIO 43976. Particularly, the high-efficiency biodegradation in the ocean or salt-containing environment is realized. The pretreatment-strain synergistic technology provided by the invention provides an efficient and green solution for pollution treatment of the waste PET in the ocean or high-salt environment, and has important application potential in a biotechnology path of plastic cyclic regeneration.

Description

TECHNICAL FIELD

[0001] The present application belongs to the fields of microorganism, high polymer material technology and organic waste biological treatment, and specifically relates to a biodegradation system for polyethylene terephthalate (PET) waste, which comprises a PET pretreatment method and functional microorganisms. The core of the system comprises a new PET-degrading bacterial strain from the ocean and a pretreatment method for reducing the crystallinity of PET by high-temperature melting-low-temperature quenching to enhance the degradation efficiency of the strain, which is particularly suitable for PET pollution treatment in salt-containing or marine environments. BACKGROUND

[0002] With the continuous expansion of global chemical industry and consumer demand, plastic products have deeply penetrated into many fields such as packaging, catering, medical treatment and textile due to their advantages of light weight, durability, low cost and convenient use. According to the Nature Communications global plastic annual production, it increased from 2 million tons in 1950 to 400 million tons in 2022, with an annual growth rate of 8.4%. Among them, polyethylene terephthalate (PET) as the core raw material for packaging beverage and food containers has a huge global annual production and increasing consumption. However, due to the imperfect recycling system and limited treatment methods, a large amount of PET products enter the natural environment after use, causing serious pollution problems.

[0003] As the final "sink" of land plastic waste, the ocean bears an increasingly heavy ecological pressure. According to the 2025 report of the United Nations Environment Programme (UNEP), it is estimated that more than 11 million tons of plastic waste flows into the ocean every year through river input, coastal activities, marine operations and other ways, not only causing ecological and economic losses, but also posing a multi-dimensional threat to the marine ecosystem. Under the action of natural factors such as ultraviolet radiation, wave impact and temperature change, the plastic entering the marine environment will gradually break and become brittle to form microplastics with a particle size of less than 5 mm. Due to the small particle size, wide distribution and strong adsorption capacity, these microplastics are easily ingested by different trophic level marine organisms such as plankton, fish and shellfish. Some organisms may cause digestive tract obstruction, malnutrition or even death due to the ingestion of microplastics. At the same time, the surface of microplastics is easy to enrich persistent organic pollutants (such as polychlorinated biphenyls), antibiotics and pathogenic bacteria, which are transmitted through the food chain and eventually pose a potential risk to human health.

[0004] From the perspective of material science, PET has a highly regular molecular structure, strong crystallinity, and is rich in stable benzene rings and ester bonds, making it extremely stable in the natural environment with a degradation period of hundreds of years. Currently, physical and chemical degradation methods generally have high cost, high energy consumption and easy secondary pollution. In contrast, biodegradation is considered the most ideal way to treat plastic pollution due to its mild conditions, environmental friendliness and no secondary pollution. However, the existing reported PET-degrading microorganisms (such asIdeonella sakaiensis 201-F6 is mostly derived from terrestrial or composting environments. Its degradation efficiency is often significantly reduced in real marine environments, especially under pressures such as high salinity, low temperature, and competition from complex microbial communities, which greatly limits its practical application.

[0005] Coastal wetland ecosystems (such as seagrass beds and mangroves), as transitional zones between the ocean and land, not only serve as collection points for plastics entering the watershed but also harbor rich and unique microbial resources. These native microorganisms have long been adapted to saline environments and possess natural degradation potential for plastics attached to their habitats, making them a valuable germplasm resource for discovering highly efficient, halophilic PET-degrading strains. Therefore, isolating and identifying new strains from these ecosystems that can efficiently degrade PET and are naturally adapted to marine environments, and developing compatible pretreatment methods that can effectively disrupt the PET crystalline structure, to form a comprehensive PET biodegradation technology system suitable for marine and high-salt environments, has become an urgent technological need for solving marine plastic pollution and realizing the resource utilization of plastic waste. Summary of the Invention

[0006] This invention provides a slow-growing rhizobium bacterium derived from a marine ecosystem with highly efficient PET degradation capabilities. Mesorhizobium sp. SCSIO 43976, along with a compatible high-temperature melting-low-temperature quenching pretreatment method that effectively reduces the crystallinity of PET, and a synergistic degradation system consisting of the two, ultimately achieve efficient biodegradation of waste PET, especially in marine or saline environments.

[0007] The first objective of this invention is to provide a slow-growing rhizobium strain isolated from a marine environment that has the function of degrading PET. Mesorhizobium sp. SCSIO 43976, deposited on October 24, 2025, at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), address: No. 100, Xianlie Middle Road, Yuexiu District, Guangzhou, Guangdong Province, 510070, China, accession number GDMCC NO: 67156. This strain can grow and metabolize using PET as its sole carbon and energy source, and is particularly suitable for degrading plastics in saline environments (such as marine habitats).

[0008] The second objective of this invention is to provide a method for degrading plastics, which utilizes the medium-slow root nodule bacterium SCSIO43976 to degrade PET.

[0009] Preferably, the plastic is PET, including but not limited to low-crystallinity PET.

[0010] Preferably, the PET is pretreated PET, which is obtained by high-temperature melting and low-temperature rapid quenching to obtain PET with low crystallinity, thereby improving its biodegradability.

[0011] Preferably, the low-temperature rapid quenching includes, but is not limited to, ice-water quenching.

[0012] A third objective of this invention is to provide the application of the medium-slow root nodule SCSIO 43976 in degrading plastics, particularly PET materials, especially in the degradation of low-crystallinity PET materials treated by the aforementioned pretreatment method.

[0013] Preferably, the application is carried out in a saline or marine environment.

[0014] Compared with the prior art, the present invention has the following significant advantages: the slow-growing rhizobium provided by the present invention Mesorhizobium sp. SCSIO 43976 can grow using waste PET as the sole carbon source. Through a developed melt-rapid cooling pretreatment method, the crystallinity of PET can be significantly reduced to 7.05%–9.29%, effectively improving the bioavailability of the material. Experimental results show that this strain can cause significant erosion morphology in PET materials, and weight loss measurements confirm its substantial degradation capability. This "pretreatment-degrading bacteria" synergistic system is suitable for marine and high-salinity environments, providing an efficient and environmentally friendly technical path for the green biodegradation of waste PET, and has significant application value for the remediation of marine plastic pollution.

[0015] Bradyrhizobia Mesorhizobium sp. SCSIO 43976 was deposited on October 24, 2025 at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), located at No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou, Guangdong Province, 510070, China, with accession number GDMCC NO: 67156. Attached Figure Description

[0016] Figure 1 This invention is a slow-growing rhizobium. Mesorhizobium Phylogenetic tree of 16S rRNA of sp. SCSIO 43976.

[0017] Figure 2 Differential scanning calorimetry (DSC) curves of PET samples pretreated with different melt holding times.

[0018] Figure 3 This is a bar chart comparing the weight loss rates of raw PET flakes and low-crystallinity PET samples obtained through different melt pretreatments during a 30-day biodegradation experiment.

[0019] Figure 4 These are scanning electron microscope (SEM) images comparing the surface morphology of PET samples before and after biodegradation.

[0020] Figure 5 This is a comparison of gel permeation chromatography (GPC) images of PET samples from different treatment groups after 30 days of degradation.

[0021] Figure 6 These are Fourier transform infrared (FT-IR) spectra of the control group and PET samples after 30 days of microbial degradation. Detailed Implementation

[0022] The following is a further detailed description of the present invention in conjunction with specific preferred embodiments, but it should not be considered that the specific embodiments of the present invention are limited to this.

[0023] Example 1: Slow-growing rhizobia Mesorhizobium Isolation and identification of sp. SCSIO 43976

[0024] Tailai grass seagrass beds in Sanya Bay, Hainan Province Thalassia hemprichii Surface sediments were collected from the growth zone and placed in a sterile sampling bag. 10g of sediment was added to 100mL of Mineral Salts Medium (MSM): (NH4)2SO4 1g, K2HPO4 100g, KH2PO4 50g, MgSO4·7H2O 0.2g, CaCl2·2H2O 0.1g, FeSO4·7H2O 0.005g, MnSO4·H2O 0.003g, ZnSO4·7H2O 0.003g, CoSO4·7H2O 0.001g, (NH4)6MoO 24 • 0.001 g of 7H₂O, pH 8.0, 1000 mL of filtered seawater, sterilized and ready for use. Using sterile waste PET bottle flakes as the sole carbon source, 1 g was added (1 cm × 1 cm, added after sterilization). The mixture was cultured in a 250 mL Erlenmeyer flask at 28°C and 180 rpm for 30 days. After culture, the enriched solution was diluted and plated, then cultured at 28°C for 14 days. Single colonies were picked, purified by three streak plating tests, and molecular biological identification was performed to obtain a pure strain of *Syntrophus stolonifera*. Mesorhizobium sp. SCSIO 43976.

[0025] Molecular biological identification method: Genomic DNA was extracted from SCSIO 43976 using a bacterial DNA extraction kit (OMEGA, USA) and amplified using universal primers 27F and 1492R (Weisenburg et al., 1991, 16S ribosomal DNA amplification for phylogenetic study., Journal of bacteriology, 1991, 173(2):697-703). The 16S rRNA sequencing results (see SEQ ID NO.1) were searched against the Ezbiocloud database to find the closest sequence in the database. Mesorhizobium qingshengii CCBAU 33460 (T) (JQ339788) has a similarity of 97.09%, which is lower than the general threshold (98.65%) for classifying new bacterial species (Chun et al., 2018, Proposed minimal standards for the use of genome data for the taxonomy of prokaryotes. International Journal of Systematic and Evolutionary Microbiology. 68: 461-466), preliminarily proving that it is a potential new species. Mesorhizobium The phylogenetic tree of the 16S rDNA of sp. SCSIO 43976 is attached. Figure 1 The strain SCSIO 43976 was named *Synchronous-slow rhizobium*. Mesorhizobium sp. SCSIO43976 was deposited on October 24, 2025 at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), located at No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou, Guangdong Province, 510070, China, with accession number GDMCC NO: 67156.

[0026] Example 2: Pretreatment method for reducing the crystallinity of waste PET bottle flakes

[0027] Cleaning: Take 7-8 waste PET bottles, remove the caps and labels, cut off the bottle body and cut it into 1cm×1cm squares. Place the resulting samples in an ultrasonic cleaner for 15 minutes, then transfer them to a 35℃ oven to dry for 2 hours until constant weight is achieved before use.

[0028] Melting: The PET sample obtained above is subjected to melt-cold quenching treatment; the temperature is set to 290℃ and preheated for 10 min until the temperature stabilizes; 2.0 g of PET sheet sample is weighed and placed in the sample tank, and kept in the molten state for 5 min, 10 min and 20 min respectively; after the melting process is completed, the sample is immediately transferred to a heat-insulating container containing an ice-water mixture and rapidly cooled for 5 min until the sample is completely solidified.

[0029] Crystallinity determination: 3.0 mg of the cooled sample was tested for crystallinity using differential scanning calorimetry (DSC) to detect the change in crystallinity of PET waste plastic bottles before and after pretreatment melt quenching. Instrument conditions: heating / cooling rate 5℃ / min, temperature range 30-300℃, nitrogen gas filling. Differential scanning calorimetry (DSC) curves of PET samples pretreated with different melt holding times (… Figure 2 Based on the enthalpy of fusion (140.1 J / g) of the PET complete crystallization theory, the crystallinity of the sample before and after treatment was calculated.

[0030] The method for calculating crystallinity is as follows:

[0031] The results showed that the crystallinity of the samples held in the molten state for 5 min, 10 min, and 20 min was 7.05%, respectively. Figure 2 B), 7.93% Figure 2 C) and 9.29% Figure 2 D), where Figure 2 A is the original film control without melting treatment.

[0032] Example 3: Analysis of PET biodegradation

[0033] Experimental Design and Sample Preparation: PET samples obtained in Example 2 after incubation at 290℃ for 5 min, 10 min, and 20 min respectively were used as experimental groups, denoted as T1, T2, and T3. Untreated raw PET bottle flakes were also set up as a control group, denoted as CK. To evaluate the degradation effect of the bacterial strain, a raw bottle flakes with added bacteria group (CKJ) was added. All groups were tested in triplicate. All PET samples (1.0 g per group) were sequentially immersed in 2% SDS solution for 24 hours and then in 75% ethanol solution for 4 hours for thorough sterilization. They were then rinsed three times with sterile water and dried in a 35℃ oven for later use.

[0034] Strain activation and degradation culture: the preserved strain Mesorhizobiumsp. SCSIO 43976 was inoculated into 2216E liquid medium for activation. The cells were collected by centrifugation and washed three times with sterile water to remove residual culture medium. Finally, the cells were resuspended in 1x inorganic salt medium (MSM) to prepare the inoculated culture solution, and the OD was adjusted. 600 The culture was inoculated to 0.8. 1 mL of the above bacterial culture was inoculated into a 250 mL sterile Erlenmeyer flask containing 1 g of PET sample (containing 30 mL of MSM inorganic salt medium, with PET as the sole carbon source). All groups were placed in a shaker and incubated in the dark at 28°C and 180 rpm for 30 days. Samples were taken every 7 days to measure the absorbance of the culture at 600 nm to monitor bacterial growth dynamics.

[0035] Degradation effect assessment: After the incubation period, all PET samples were removed and subjected to surface cleaning and drying again using the initial method until constant weight was achieved. The mass after incubation was accurately weighed, and the weight loss rate was calculated using the following formula.

[0036] Weight loss rate (%) = ×100%.

[0037] Based on the above formula, the weight loss rates of raw PET flakes and low-crystallinity PET samples obtained through different melt pretreatments were calculated during a 30-day biodegradation experiment. The comparison bar chart is shown below. Figure 3 As shown, the average weight loss rate for each group is as follows:

[0038] CK group (unprocessed original film): 0.046% 0.034%

[0039] CKJ group (untreated original slide with added bacteria): 0.080% 0.0302%

[0040] Group T1 (holding temperature for 5 min): 1.114% 1.35%

[0041] Group T2 (insulated for 10 min): 0.315% 0.0511%

[0042] Group T3 (insulated for 20 min): 0.510% 0.453%

[0043] The results showed that the degradation experimental data fully verified the superior effectiveness of the technical solution of this invention. Specifically, the degradation rate (1.114%) of the T1 group sample, after 5 minutes of pretreatment, was 24.2 times that of the untreated blank control group (CK) and 13.9 times that of the untreated but inoculated control group (CKJ). The synergistic technical system consisting of "short-time melt-quench pretreatment" and "dedicated degrading strain" provided by this invention provides important scientific data and technical support for the efficient biodegradation of waste PET.

[0044] Surface morphology analysis of samples: After cultivation, PET samples from both the experimental and control groups were removed and gently rinsed three times with sterile phosphate-buffered saline (PBS, 0.1 M, pH 7.4) to remove loosely attached bacteria and culture medium residues. The samples were then fixed in 2.5% (v / v) glutaraldehyde solution at 4°C for 12 hours. After fixation, the samples were rinsed three times with the same PBS buffer, followed by a series of dehydration steps with 30%, 50%, 70%, 80%, 90%, and 100% (v / v) ethanol solutions, each soaking for 15 minutes. The cultured PET samples were then cut into 10 mm × 10 mm pieces, fixed, and sputter-coated with gold. The changes in surface micromorphology were observed using a scanning electron microscope (SEM; SU8600, Hitachi, Japan). The processed samples were observed using a scanning electron microscope (SEM; SU8600, Hitachi, Japan). The microstructure of the sample surface was observed and recorded at different magnifications (2000× to 4000×) under an accelerating voltage of 15 kV and high vacuum mode. The results are as follows: Figure 4As shown, there are significant differences in the surface morphology of PET samples between the experimental and control groups. The experimental groups (especially T1 group) exhibit significant large, irregular, blocky structures on the PET surface, with visible layering and curling at the edges. The structures are thick and clustered. The T2 group surface is dominated by small granular and sheet-like structures, with small and dispersed particles, and a continuous substrate structure visible in the background. The particles show uneven aggregation and distribution defects such as micropores between them and the substrate. The T3 group exhibits large-scale cracks and layered structures, with a macroscopic morphology and obvious "fracture surface" characteristics. In contrast, the CK group (untreated) surface has a continuous filamentous film structure, soft and uniform, with only natural wrinkles. The CKJ group (inoculated only) shows sheet-like and fragmented accumulations, a compact structure, localized fractures and cracks, and no continuous film areas. The experimental groups (especially T1 group) show significant microbial erosion morphology on the PET surface, including abundant bacterial attachment, biofilm formation, and obvious cracks and pits caused by degradation, with plastic degradation residues visible within the pits. In contrast, the control group samples had a smoother surface with only a few mechanical scratches and no obvious signs of biological erosion or degradation.

[0045] Determination of PET molecular weight and distribution: To quantitatively analyze the degree of PET degradation caused by the experimental treatment, gel permeation chromatography (GPC) was used to determine the changes in sample molecular weight. The residual PET sample after degradation was dissolved in chromatographically pure tetrahydrofuran (THF) to prepare a 1.0 mg / mL solution, which was then filtered through a 0.45 μm microporous membrane before injection. An Agilent 1260 GPC system was used with THF as the mobile phase at a flow rate of 1.00 mL / min and a column temperature of 40 °C for a run time of 40 minutes. The system was calibrated with narrow-distribution polystyrene standards.

[0046] GPC measurement results show that ( Figure 5 (See Table 1) Compared with the control group (CK and CKJ), the peak molecular weight (Mp), number-average molecular weight (Mn), and weight-average molecular weight (Mw) of the experimental group (T1, T2, T3) PET samples all decreased significantly, and the polydispersity index (PD) decreased simultaneously.

[0047] Table 1. Molecular weight and distribution of PET samples after degradation in different treatment groups

[0048]

[0049] Group T1: The ratio of Mn to Mp is (22860 / 41938)×100%≈54.5%;

[0050] Group T2: The ratio of Mn to Mp is (18986 / 33762)×100%≈56.2%;

[0051] Group T3: The ratio of Mn to Mp is (21100 / 34209)×100%≈61.7%.

[0052] GPC analysis showed that the molecular weight of PET samples in all experimental groups was significantly reduced. Group T1 had the lowest Mn / Mp ratio (54.5%), indicating the highest proportion of low molecular weight products and the most complete degradation. Although the Mn / Mp ratio of group T2 (56.2%) was lower than that of group T3 (61.7%), the higher initial Mp value of group T3 and its ability to achieve a comparable molecular weight after degradation suggest that group T3 exhibited more efficient breakage of high molecular weight segments. These results confirm at the molecular level that microbial degradation led to substantial chain breakage in PET polymers, rather than merely surface erosion.

[0053] Fourier transform infrared spectroscopy (FTIR) determination: Residual PET samples after degradation were collected and analyzed using a Fourier transform infrared spectrometer. Background correction was applied to the air, and the scanning range was set to 4000–400 cm⁻¹. The samples were then placed on a test stage illuminated by the light beam and gently compressed to ensure proper probe illumination. Finally, the instrument was started to acquire the infrared spectral data. Each sample was measured in triplicate, and the average spectrum was obtained. Results are as follows: Figure 6 As shown, the transmittance of the CK and CKJ groups in the 700-1713 cm⁻¹ range was significantly lower than that of the experimental groups (T1, T2, T3). This phenomenon indicates that the PET polymer chain structure in the control group was intact, with functional groups concentrated in the macromolecular backbone, resulting in strong absorption of infrared light and thus lower transmittance. In contrast, the experimental group underwent effective degradation, with the long PET chains breaking into smaller molecular fragments. The same functional groups were distributed in more independent molecules, increasing spatial dispersion and relatively weakening the overall absorption intensity, leading to higher transmittance. This result confirms at the chemical structure level that the PET in the experimental group underwent substantial molecular chain breakage.

[0054] 16S rDNA

[0055] > Mesorhizobium .sp SCSIO43976 (SEQ ID NO.1)

[0056]

Claims

1. Mesorhizobium mesophora Mesorhizobium sp. SCSIO 43976, accession number GDMCC NO: 67156.

2. A method for degrading plastics, characterized in that, The method utilizes the slow-growing rhizobium SCSIO43976 described in claim 1 to degrade PET.

3. The method according to claim 2, characterized in that, The plastic mentioned is PET.

4. The method according to claim 3, characterized in that, The PET mentioned includes, but is not limited to, low-crystallinity PET.

5. The method according to claim 3, characterized in that, The PET described is obtained by high-temperature melting followed by low-temperature rapid quenching to achieve low crystallinity.

6. The method according to claim 5, characterized in that, The aforementioned low-temperature rapid quenching includes, but is not limited to, ice-water quenching.

7. The application of the slow-growing rhizobium SCSIO 43976 as described in claim 1 in the degradation of plastics.

8. The application according to claim 7, characterized in that, The plastic mentioned is PET material.

9. The application according to claim 8, characterized in that, The PET material mentioned is a pretreated, low-crystallinity PET material.

10. The application according to claim 7, characterized in that, The application is carried out in saline or marine environments.

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