Combined flora YB2 capable of degrading PET (Polyethylene Terephthalate) plastic and application of combined flora YB2
Through the combined bacteria YB2 composed of onion Berkholderia ZY1 and Pseudomonas Harudini G1B, the problem of difficult degradation of PET plastics is solved, and the PET film and intermediate products are efficiently degraded, which significantly improves environmental pollution.
Patent Information
- Application Number
- CN202510488525.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-18
- Publication Date
- 2025-07-25
AI Technical Summary
In the prior art, PET plastics are difficult to effectively degrade, resulting in environmental pollution and health threats, the degradation efficiency of a single strain is low, and the synergy of microbial flora is not fully utilized.
The combined bacteria YB2 composed of Burkholderia onion ZY1 and Pseudomonas Harudini G1B was adopted. The optimal inoculation ratio was 4:5. PET plastics and intermediate products BHET, MHET and TPA were degraded at pH 7.0 and 30°C.
The 4.3% weight loss rate of the PET film was achieved, and obvious cracks and pores appeared on the surface of the PET film, and the key functional groups C-O, C=O and C-H were reduced, which significantly improved the PET degradation efficiency.
Smart Images
Figure CN120366122A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to a consortium YB2 capable of degrading PET plastics and its application, belonging to the field of microbial technology. Background Art
[0002] PET is one of the most commonly used plastics at present. Due to its good wear resistance, impact resistance and light weight, it is widely used in food packaging, textile industry, chemical industry and other fields. With the continuous increase in the demand for PET, PET waste is widespread in the environment. However, due to the complex structure of PET and its difficulty in degradation, it can exist in nature for a long time, not only causing serious environmental pollution, but also posing a potential threat to organisms and human health. Therefore, how to effectively reduce PET pollution has become a global focus. The PET biodegradation method is considered to be one of the most promising degradation methods due to its simple operation, environmental friendliness, low energy consumption and other advantages. At present, the number of strains with PET degradation ability is limited and the degradation efficiency is low. Compared with the degradation of PET by a single strain, microbial consortia can have a higher degradation efficiency for PET through the synergistic effect between strains. It can be seen that microbial consortia have opened up a new way for the biodegradation of PET. Summary of the Invention
[0003] The purpose of the present invention is to provide a consortium YB2 capable of degrading PET plastics and its application. The combined degradation system is composed of two non-antagonistic Burkholderia cepacia ZY1 and Pseudomonas halodini G1B.
[0004] In order to achieve the above purpose, the present invention adopts the following technical scheme: A consortium YB2 capable of degrading PET plastics, the consortium YB2 is composed of Burkholderia cepacia ZY1 and Pseudomonas halodini G1B. The Burkholderia cepacia ZY1 is preserved in the China Center for Type Culture Collection, with the preservation number CCTCC NO: M 20232644, and the preservation date is December 22, 2023; Pseudomonas halodini G1B is preserved in the China General Microbiological Culture Collection Center, with the preservation number CGMCC NO: 31904, and the preservation date is September 9, 2024.
[0005] The preparation method of the above-mentioned consortium YB2 includes the following steps: (1) Inoculate Burkholderia cepacia ZY1 and Pseudomonas halodini G1B into LB liquid medium respectively, and culture them overnight in a shaker at 30 °C to obtain the activated strains; (2) Pick the two activated single bacteria and inoculate them into LB liquid medium respectively, and culture them until the OD600 is 0.8.
[0006] (3) Mix Burkholderia cepacia ZY1 and Pseudomonas halodurans G1B at an inoculation amount ratio of 4%:5% to obtain a combined microbial community YB2.
[0007] The present invention also provides a microbial agent capable of degrading PET plastics, which contains the above-mentioned combined microbial community YB2.
[0008] The above-mentioned combined microbial community YB2 or the above-mentioned microbial agent can be applied to degrade PET plastics and their intermediate products.
[0009] The present invention also provides a method for degrading PET plastics. The steps are to inoculate the above-mentioned combined microbial community YB2 into a liquid inorganic salt medium containing PET film and its intermediate products respectively for cultivation.
[0010] Further, the inoculation ratio of Burkholderia cepacia ZY1 to Pseudomonas halodurans G1B is 4%:5%.
[0011] Further, the concentration of the PET film is 1 g / L.
[0012] Further, the intermediate products are BHET, MHET, and TPA.
[0013] Further, the concentrations of the intermediate products BHET, MHET, and TPA are all 100 mg / L.
[0014] The beneficial effects of the present invention are as follows: The present invention provides a combined microbial community YB2 capable of degrading PET plastics and its application. The combined microbial community YB2 is composed of Burkholderia cepacia ZY1 and Pseudomonas halodurans G1B. Their optimal inoculation ratio is 4:5, and the optimal growth conditions are pH 7.0 and 30°C. The combined microbial community YB2 can not only effectively degrade PET films, but also effectively degrade the PET intermediate products BHET, MHET, and TPA. The weight loss rate of the PET film treated by the combined microbial community YB2 is 4.3%. There are obvious cracks and pores on the surface of the PET film, and the key functional groups of C-O, C=O, and C-H on the PET film are all significantly reduced. Description of the Drawings
[0015] Figure 1 Antagonistic experiment diagram of strain ZY1 and strain G1B.
[0016] Figure 2 Optimal growth conditions of the combined microbial community YB2.
[0017] Figure 3 LC-MS analysis diagram of the degradation of PET by the combined microbial community YB2.
[0018] Figure 4 Degradation of PET intermediate products by the consortium YB2
[0019] Figure 5 SEM image of the PET membrane treated with the consortium YB2
[0020] Figure 6 FTIR image of the PET membrane treated with the consortium YB2 Detailed implementation manners
[0021] In order to make the content of the present invention easier to understand, the technical solutions of the present invention will be further described below in conjunction with specific implementation manners, but the present invention is not limited thereto only.
[0022] The polyethylene terephthalate (PET) film in the following examples was purchased from Guangyuan Plasticizing Company.
[0023] LB liquid medium: NaCl 10 g / L, tryptone 10 g / L, yeast extract 5 g / L, sterilized by high-temperature steam at 121 °C for 15 min and then reserved for use. for 15 min and then reserved for use.
[0024] Inorganic salt liquid medium (MSM liquid medium): K2PO4 1.5 g / L, KH2PO4 0.5 g / L, MgSO4·7H2O 0.2 g / L, (NH4)2SO4 1.5 g / L, NaCl 0.5 g / L, FeCl3·6H2O 0.001 g / L, CaCl2·2H2O 0.02 g / L, sterilized by high-temperature steam at 121 °C for 15 min and then reserved for use.
[0025] Unless otherwise specified, ZY1 mentioned in the following examples is Burkholderia cepacia ZY1, which is preserved in the China Center for Type Culture Collection, with the preservation number CCTCC NO: M 20232644 and the preservation date of December 22, 2023; G1B mentioned in the following examples is Pseudomonas halodini G1B, which is preserved in the China General Microbiological Culture Collection Center, with the preservation number CGMCC NO: 31904 and the preservation date of September 9, 2024.
[0026] 1 Experimental method 1.1 Confrontation experiment between strain ZY1 and strain G1B Take 50 μL of the preserved ZY1 and G1B bacterial solutions respectively and culture them overnight in 5 mL of LB liquid medium; spread the activated G1B bacterial solution on an LB plate. After the colonies grow, select colonies with uniform growth trends, punch holes to prepare bacterial cakes; inoculate the activated ZY1 bacterial solution into 2 mL of LB liquid medium. When the OD600 reaches about 1, take 100 μL of the ZY1 bacterial solution and spread it evenly on the solid LB medium. Then inoculate the pre-made G1B bacterial cake on the LB plate coated with the ZY1 bacterial solution, and inoculate it at 4 symmetric points 2 cm away from the center of the bacterial cake respectively.
[0027] 1.2 Construction of the combined bacterial community of strains ZY1 and G1B (1) Inoculate Burkholderia cepacia ZY1 and Pseudomonas halodurans G1B into LB liquid medium respectively, and culture them overnight in a shaker at 30 °C to obtain activated strains; (2) Pick the two activated single bacteria and inoculate them into LB liquid medium respectively, and culture them until the OD600 is about 0.8.
[0028] (3) Mix Burkholderia cepacia ZY1 and Pseudomonas halodurans G1B at an inoculation amount of 4%:5% to obtain the activated combined bacterial community YB2.
[0029] 1.3 Optimal growth conditions of the combined bacterial community YB2 Inoculate the activated combined bacterial community YB2 into LB liquid medium adjusted to pH 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, and 11.0 with HCl or NaOH for culture. Culture it with shaking at 30 °C and 180 rpm, set 3 parallels, sample every 6 h, and use an ultraviolet spectrophotometer to detect the absorbance value at 600 nm of the culture solution. Similarly, culture it at pH 7 and different temperatures (20 °C, 25 °C, 30 °C, 35 °C, 40 °C), set 3 parallels for each, sample every 6 h, and use an ultraviolet spectrophotometer to detect the absorbance value at 600 nm of the culture solution.
[0030] 1.4 LC-MS analysis of the degradation of PET by the combined bacterial community YB2 Inoculate the activated combined bacterial community YB2 into the inorganic salt liquid medium (MSM liquid medium) containing 1 g / L PET film, set 3 parallel groups, and use the MSM liquid medium without bacteria as the control group. After culturing in the dark at 30 °C and 180 rpm for 3 d, sample for LC-MS detection.
[0031] 1.5 Degradation of PET intermediate products by the combined bacterial community YB2 The activated combined bacterial community YB2 was inoculated into MSM liquid media containing BHET, MHET, and TPA at a concentration of 100 mg / L each. Three parallel groups were set up, and samples were taken at 3 d, 5 d, and 7 d to measure the OD 600 value and detect the residual amounts of BHET, MHET, and TPA using HPLC.
[0032] 1.6 Analysis of the weight loss rate of PET film by the combined bacterial community YB2 The PET film treated with the combined bacterial community YB2 for 3 d was washed 3 - 5 times with deionized water, placed in a sterile centrifuge tube containing 30 mL of 2% SDS solution, shaken at 37 ºC and 230 rpm for 2 h, placed in an ultrasonic cleaner for 30 min, then washed several times with 70% absolute ethanol, and finally dried in an oven at 100 ºC and weighed. The washed PET film was weighed.
[0033] 1.7 Analysis of the surface damage of PET film by the combined bacterial community YB2 The PET film treated with the combined bacterial community YB2 was washed 3 - 5 times with deionized water, placed in a sterile centrifuge tube containing 30 mL of 2% SDS solution, shaken at 37 ºC and 230 rpm for 2 h, placed in an ultrasonic cleaner for 30 min, then washed several times with 70% absolute ethanol, and finally dried in an oven at 100 ºC, with the untreated PET film as a control. A conductive adhesive strip was smeared on the sample stage, the PET sample was picked up with tweezers and stuck to the conductive adhesive strip, and the sample stage with the PET sample was placed in an ion sputtering instrument. After sputtering gold for 60 s under vacuum, it was placed under a scanning electron microscope (SEM) to adjust the magnification and focus to observe its microscopic morphological characteristics. After determination, the scanning image was taken and saved.
[0034] 1.8 Analysis of the change in surface functional groups of PET film by the combined bacterial community YB2 The PET film treated with the combined bacterial community YB2 was washed 3 - 5 times with deionized water, placed in a sterile centrifuge tube containing 30 mL of 2% SDS solution, shaken at 37 ºC and 230 rpm for 2 h, placed in an ultrasonic cleaner for 30 min, then washed several times with 70% absolute ethanol, and finally dried in an oven at 100 ºC, with the untreated PET film as a control. Fourier transform infrared spectroscopy (FTIR) was used to characterize the change in surface functional groups of PET, and the ATR mode was used. The spectrum of 400 - 4000 cm -1 was obtained using OMNIC software and scanned 32 times on average.
[0035] 2 Results and analysis 2.1 Confrontation experiment between strain ZY1 and strain G1B The results of the confrontation experiment between strain ZY1 and strain G1B are as Figure 1 shown. There was no sterile zone formed around the G1B bacterial cake, indicating that there was no mutual antagonism between this strain and the spread ZY1 strain.
[0036] 2.2 Optimal growth conditions of the consortium YB2 The growth of the consortium YB2 at different pH values is as Figure 2 shown in (a). When the pH was 4 - 8, the OD 600 value of the consortium YB2 showed a trend of first increasing and then decreasing with the extension of time. Among them, when the pH was 7, the OD 600 value of the consortium YB2 was overall higher than the OD 600 values at other pH values, and the OD 600 reached the maximum value at 24 h, which was 6.50. When the pH was 4, 5, 6, and 8, the OD 600 values of the consortium YB2 all reached the maximum value at the 24th h. When the pH was 10 and 11, the OD 600 values of the consortium YB2 were extremely low, remaining between 0.10 - 0.12 all the time, indicating that the consortium YB2 could not grow in a strongly alkaline environment. In summary, the optimal growth pH of the consortium YB2 is 7.
[0037] The growth of the consortium YB2 at different temperatures is as Figure 2 shown in (b). Overall, the OD 600 values of the consortium YB2 at different temperatures all showed a trend of first increasing and then decreasing with the extension of time, and the OD 600 reached the highest value at the 24th h. Their growth rates and biomass were 30ºC > 35ºC > 40ºC > 25ºC > 20ºC. At 30ºC, the growth state of the consortium YB2 was the most active, and the growth state of the consortium YB2 reached its peak at 24 h, with the OD 600 value being 6.20. In summary, the optimal growth temperature of the consortium YB2 is 30ºC.
[0038] 2.3 LC - MS analysis of the PET degradation by the consortium YB2 Through LC - MS analysis of the PET degradation products of the consortium YB2, the results are as Figure 3 shown. Four ion fragment peaks of PET metabolites were detected in the supernatant of the consortium YB2 culture. The ion fragments of BHET and diisobutyl terephthalate were detected in the positive ion mode ( Figure 3 (c) and (f)); in the negative ion mode, the ion fragments of MHET and TPA were detected ( Figure 3((d) and (e)). In summary, the degradation products of the combined microbial community YB2 on PET are BHET, MHET, TPA, and diisobutyl terephthalate.
[0039] 2.4 Degradation of PET intermediate products by the combined microbial community YB2 The degradation of PET intermediate products BHET, MHET, and TPA by the combined microbial community YB2 is as follows Figure 4 shown. During the entire culture process, the growth rate and OD 600 value of the combined microbial community YB2 in the culture solution containing BHET and MHET are relatively close, and are significantly less than the growth rate and OD 600 value in the culture solution containing TPA. In the first 2 days, the degradation rate and OD 600 value of the combined microbial community YB2 for the three intermediate products gradually increased with the prolongation of time. Especially on the 1st day, the combined microbial community YB2 rapidly degraded the intermediate products, and the OD 600 value also increased rapidly. On the 2nd day, the degradation rates reached 96.4%, 97.3%, and 88.3% respectively, and the OD 600 values were 1.30, 1.15, and 4.01 respectively. After the 2nd day, the degradation rates of the three intermediate products gradually leveled off, and the OD 600 also gradually leveled off. By the 5th day, the degradation rates of the combined microbial community YB2 for the three intermediate products all reached over 97%.
[0040] 2.5 Analysis of the weight loss rate of PET film by the combined microbial community YB2 The degradation effect on PET was analyzed by the weight change of the PET film before and after treatment with the combined microbial community YB2. It was experimentally obtained that the weight of the PET film decreased by 4.3% after being treated with the combined microbial community YB2 for 3 days.
[0041] 2.6 Analysis of the surface damage of PET film by the combined microbial community YB2 The surface damage of the PET film after treatment with the combined microbial community YB2 is as follows Figure 5 shown. At 600 times, 1000 times, and 5000 times, the surface of the PET film without any treatment is flat, smooth, and unchanged ( Figure 5 (a)-(c)), and the surface of the PET film after treatment with the combined microbial community YB2 is rough with folds, obvious cracks and holes, and many degraded fragments adhere to the surface of the PET film ( Figure 5 (d)-f)), further proving that the combined microbial community YB2 has high degradation activity on the PET film.
[0042] 2.7 Analysis of the change of surface functional groups of PET film by the combined microbial community YB2 The FTIR results of the PET film are as follows Figure 6As shown, compared with the PET film without any treatment, the PET film treated with the combined flora YB2 shows a decrease in the absorption peak intensity of several key functional groups, including C-H at 730 cm -1 , C-O at 1050 cm -1 , 1094 cm -1 , and 1240 cm -1 , and C=O between 1700 - 1750 cm -1 . This indicates that the combined flora YB2 mainly degrades PET by breaking alkoxy groups and carbonyl groups, resulting in a decrease in oxygen content.
[0043] Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Any person familiar with this technology can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.
Claims
1. A consortium of bacteria YB2 capable of degrading PET plastics, characterized in that: The combined bacterial community YB2 is composed of Burkholderia cepacia ZY1 and Pseudomonas halodini G1B. Burkholderia cepacia ZY1 is deposited in the China Center for Type Culture Collection with the deposit number CCTCC NO: M 20232644 and the deposit date of December 22, 2023; Pseudomonas halodini G1B is deposited in the China General Microbiological Culture Collection Center with the deposit number CGMCC NO: 31904 and the deposit date of September 9, 2024.
2. The preparation method of the combined flora YB2 according to claim 1, characterized in that : The method includes the following steps: (1) Inoculate Burkholderia cepacia ZY1 and Pseudomonas halodini G1B separately into LB liquid medium and culture overnight in a shaker at 30 °C to obtain activated single strains. (2) Pick the two activated single strains and inoculate them separately into LB liquid medium, and culture them until the OD600 reaches 0.
8. (3) Mix Burkholderia cepacia ZY1 and Pseudomonas halodini G1B with an inoculation amount ratio of 4%:5% to obtain the combined bacterial community YB2.
3. A bacterial agent for degrading PET plastics, characterized in that: It contains the combined bacterial community YB2 described in claim 1.
4. Application of the combined bacterial community YB2 described in claim 1 or the bacterial agent described in claim 3 in degrading PET plastics and their intermediate products.
5. A method for degrading PET plastics, characterized in that: Inoculate the combined bacterial community YB2 described in claim 1 separately into a liquid inorganic salt medium containing PET film and its intermediate products for culture.
6. The application according to claim 5, wherein: The inoculation ratio of Burkholderia cepacia ZY1 and Pseudomonas halodini G1B is 4%:5%.
7. The method according to claim 5, wherein: The concentration of the PET film is 1 g / L.
8. The method according to claim 5, wherein: The intermediate products are BHET, MHET, and TPA.
9. The method according to claim 8, characterized in that: The concentrations of the intermediate products BHET, MHET, and TPA are all 100 mg / L.
Citation Information
Cited By
Burkholderia cepacia-sourced esterase as well as coding gene and application thereof
CN121518434A