Pseudomonas mutabilis and application thereof
By providing a Pseudomonas morphogen YL3 that can degrade PET plastics, the problem of difficulty in degrading PET plastics is solved, and the effective degradation of PET plastics is achieved, and the potential of industrial biodegrading plastics is possessed.
Patent Information
- Application Number
- CN202411872198.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-18
- Publication Date
- 2025-02-07
AI Technical Summary
The prior art is difficult to effectively degrade polyethylene terephthalate (PET) plastics, making it difficult for PET plastics to degrade in nature and become the main source of white pollution.
A strain of Pseudomonas plecoglossicida YL3 was provided, which can effectively degrade PET plastic and its intermediate degradation products, and degrade at 30°C by contacting PET plastic.
This strain was able to significantly degrade PET plastic, the weight loss rate of PET film reached 3.43%, and cracks and pores formed on the surface, and the ester bond functional groups were reduced, demonstrating its ability to effectively degrade PET.
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Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of microorganisms, and in particular relates to a strain of Pseudomonas proteus and an application thereof. Background Art
[0002] Polyester plastic polyethylene terephthalate (PET) is one of the most commonly used petroleum-based plastics in the world. It is a high molecular polymer formed by connecting terephthalic acid and ethylene glycol through ester bonds. PET is widely used in many fields such as food, packaging, electronic products and textiles due to its excellent plasticity. In recent years, PET plastics have been particularly popular in the field of food packaging. For example, heat-resistant bottled beverages such as tea and juice all use PET plastic external packaging technology. At present, due to the widespread use of PET, PET solid waste generated each year has accounted for 12% of the world's solid waste. Most PET products have a high degree of crystallinity and are durable against mechanical and chemical stresses. After being discarded, they are extremely difficult to degrade in nature, thus becoming the main source of white pollution. Therefore, how to effectively deal with PET has become a hot topic of social concern.
[0003] At present, the methods for processing PET plastics mainly include physical methods, chemical methods and biological methods. Processing PET by physical and chemical methods requires a large amount of material energy, and there is a risk of polluting the environment and destroying the ecology. Compared with physical and chemical methods, the biodegradation method of PET has the advantages of easy operation, environmental friendliness and low energy consumption, and is currently the most environmentally friendly method. Therefore, the PET biodegradation method is considered to be the most promising and potential method. At present, the biodegradation method mainly utilizes microbial degradation. At present, the microorganisms that can degrade PET mainly include Vibrio, Bacillus, Pseudomonas, Thermospora and other microorganisms.
[0004] Pseudomonas belongs to the phylum Gammaproteobacteria, which is the most common bacterial phylum involved in plastic degradation. It is reported that Gammaproteobacteria can degrade 43 of the 66 reported plastics, including most of the mass-produced synthetic plastics such as PE, PET, PP, PS, PVC and PUR. Pseudomonas can degrade 35 different types of plastics. Pseudomonas is highly adaptable, widely used and ubiquitous in the environment. These data highlight its importance and potential for plastic degradation. The recent report on the bacterium Pseudomonas plecoglossicida is about the degradation of the environmental pollutant cyclohexylamine, and it has not been confirmed to have the ability to degrade plastics. Summary of the invention
[0005] The purpose of the present invention is to provide a strain of Pseudomonas proteus and its application. The Pseudomonas proteus provided by the present invention can effectively degrade PET, can be used as a microorganism for PET biodegradation, enriches the bacterial species resources for PET biodegradation, and has broad application prospects.
[0006] The technical solution adopted by the present invention is as follows: The first aspect of the present invention provides a strain of Pseudomonas plecoglossicida, which is named Pseudomonas plecoglossicida YL3, and was deposited in the General Microbiology Center of China National Microbiological Culture Collection Committee on September 9, 2024, with a deposit address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and a deposit number of CGMCC NO.31905.
[0007] The second aspect of the present invention provides the use of the above-mentioned strain of Pseudomonas proteus in degrading polyethylene terephthalate and its intermediate degradation products; Furthermore, the intermediate degradation products of polyethylene terephthalate include dihydroxyethyl terephthalate, ethylene terephthalate, and terephthalic acid.
[0008] The third aspect of the present invention provides the use of the above-mentioned strain of Pseudomonas proteus in preparing a bacterial agent for degrading polyethylene terephthalate and its intermediate degradation products.
[0009] The fourth aspect of the present invention provides a bacterial agent, which contains the above-mentioned Pseudomonas proteus.
[0010] The fifth aspect of the present invention provides the use of the above-mentioned bacterial agent in degrading polyethylene terephthalate and intermediate degradation products of polyethylene terephthalate; Furthermore, the intermediate degradation products of polyethylene terephthalate include dihydroxyethyl terephthalate, ethylene terephthalate, and terephthalic acid.
[0011] The sixth aspect of the present invention provides a method for degrading polyethylene terephthalate, which comprises contacting the above-mentioned Pseudomonas proteus or bacterial agent with polyethylene terephthalate and performing degradation at 30°C.
[0012] The seventh aspect of the present invention provides a method for degrading an intermediate degradation product of polyethylene terephthalate, which comprises contacting the above-mentioned Pseudomonas proteus or bacterial agent with the intermediate degradation product of polyethylene terephthalate and degrading the intermediate degradation product at 30°C; Furthermore, the intermediate degradation products of polyethylene terephthalate include dihydroxyethyl terephthalate, ethylene terephthalate, and terephthalic acid.
[0013] The significant advantages of the present invention are: The present invention provides a strain of Pseudomonas proteus YL3, which is reported for the first time to have the ability to degrade PET and can effectively degrade PET intermediate degradation products BHET, MHET and TPA. The weight loss rate of a PET film treated with the strain for 11 days is as high as 3.43%, and there are obvious cracks and pores on the surface of the PET film, and the ester bond functional groups on the surface of the PET film are reduced. The present invention uses the Pseudomonas proteus YL3 for PET degradation, saves production costs, has the potential for industrial biodegradation of plastics, and enriches the resources of PET degradation strains. BRIEF DESCRIPTION OF THE DRAWINGS
[0014] Figure 1 : Colony morphology and electron micrographs of Pseudomonas aeruginosa YL3.
[0015] Figure 2 : Phylogenetic tree diagram of Pseudomonas aeruginosa YL3.
[0016] Figure 3 : HPLC graph of PET film degradation by Pseudomonas aeruginosa YL3.
[0017] Figure 4 : SEM images of Pseudomonas proteus YL3 colonized on PET film.
[0018] Figure 5 : SEM images of PET film treated with Pseudomonas proteus YL3.
[0019] Figure 6 : FTIR images of PET film treated with Pseudomonas proteus YL3.
[0020] Figure 7 : Degradation curves of intermediate degradation products of PET by Pseudomonas proteus YL3. DETAILED DESCRIPTION
[0021] In order to make the contents of the present invention easier to understand, the technical solution of the present invention is further described below in conjunction with specific implementation methods, but the present invention is not limited thereto.
[0022] Embodiment 1: 1 Experimental Materials and Methods 1.1 The standards involved in the examples are as follows: Polyethylene terephthalate (PET) film was purchased from Guangyuan Plastics Company, bis(hydroxyethyl) terephthalate (BHET) was purchased from Sigma Company, ethylene glycol terephthalate (MHET) was purchased from Aladdin Company, and terephthalic acid (TPA) was purchased from McLean Company.
[0023] 1.2 The culture medium involved in the embodiment is as follows: LB liquid culture medium: NaCl 10g / L, tryptone 10g / L, yeast extract 5g / L, sterilize with high-temperature steam at 121℃ for 20min before use.
[0024] LB solid culture medium: NaCl 10 g / L, tryptone 10 g / L, yeast extract 5 g / L, and 1.5 wt% agar powder, sterilized by high-temperature steam at 121°C for 20 min before use.
[0025] Inorganic salt liquid culture medium (MSM liquid culture medium): K2HPO4 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, FeCl·6H2O 0.001 g / L, CaCl·2H2O 0.02 g / L, sterilize with high-temperature steam at 1121°C for 20 min before use.
[0026] Inorganic salt solid culture medium (MSM solid culture medium): K2HPO4 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, FeCl·6H2O 0.001 g / L, CaCl·2H2O 0.02 g / L, and 1.5 wt% agar powder are added, and sterilized with high-temperature steam at 121°C for 20 min before use.
[0027] 1.3 The screening, separation and identification of PET-degrading strains involved in the examples are as follows: (1) Using PET film as substrate, the microorganisms in the environment were subjected to five consecutive rounds of gradient enrichment and domestication: In the first round of enrichment and acclimatization, 10 g of soil sample from Yulong Snow Mountain was weighed and added to 100 mL of inorganic salt liquid culture medium containing 1 g / L PET film as the sole carbon source, and cultured at 30°C, 180 rpm and in the dark for 7 days; in the second round of enrichment and acclimatization, 10 mL of the first round of enrichment solution was added to 100 mL of fresh inorganic salt liquid culture medium containing 2 g / L PET film as the sole carbon source, and cultured at 30°C, 180 rpm and in the dark for 7 days; in the third round of enrichment, 10 mL of the second round of enrichment solution was added to 100 mL of fresh inorganic salt liquid culture medium containing 3 g / L PET film as the sole carbon source, and cultured at 30°C, 180 rpm and in the dark for 7 days; in the fourth round of enrichment, 10 mL of the third round of enrichment solution was added to 100 mL of 4 g / L PET membrane was used as the sole carbon source in a fresh inorganic salt liquid culture medium, and cultured at 30°C, 180 rpm in the dark for 7 days. In the fifth round of enrichment, 10 mL of the fourth round enrichment solution was added to 100 mL of a fresh inorganic salt liquid culture medium containing 5 g / L PET membrane as the sole carbon source, and cultured at 30°C, 180 rpm in the dark for 7 days.
[0028] (2) Separation and purification Take 200 μL of the fifth round of enrichment solution and spread it on MSM solid medium containing PET, culture it at 30°C in the dark, select well-growing colonies for streaking to isolate and purify single bacteria, and obtain the target strain YL3.
[0029] Pick a single colony of the target strain YL3 and culture it in LB liquid medium until OD 600 =0.8, add 50xol% glycerol in equal volume to the culture medium and mix well, then store it at -80°C. Before use, activate the YL3 glycerol-preserved bacteria stored at -80°C by streaking with LB solid medium, and after activation, pick a single bacterium and culture it in LB liquid medium until OD 600 =1.0, which is the bacterial suspension of strain YL3.
[0030] (3) Second screening: PET degradation activity analysis of the target strains obtained in the initial screening 1.2 mL of the bacterial suspension of target strain YL3 was taken with an inoculum volume of 3 vol% (OD 600 =1.0) to 40 mL of an inorganic salt liquid culture medium containing 1 g / L PET film, three parallel groups were set up (with no bacteria added as the control group), and after culturing in the dark at 30°C and 180 rpm for 10 days, samples were taken for HPLC determination to analyze the degradation of PET by the target strain.
[0031] (4) Molecular biological identification of PET-degrading bacteria The total DNA of strain YL3 was extracted by referring to the bacterial genome extraction kit TIANamp Bacteria DNA Kit 50. The bacterial universal primers 16S(F) (5'-AGAGTTTGSTCCTGGCTCAG-3') and 16S(R) (5'-GGTTACCTTGTTACGACTT-3') were used for 16Sr DNA amplification. The PCR used a 25μL reaction system, including 2×TaqMaster Mix 12.5μL, 16S(F) (10μmol / L) 1μL, 16S(R) (10μmol / L) 1μL, DNA template 1μL, ddH2O9.5μL. The PCR amplification program was as follows: pre-denaturation at 94℃ for 3min; denaturation at 94℃ for 30s, annealing at 50℃ for 45s, extension at 72℃ for 1min 40s, for a total of 35 cycles; and finally extension at 72℃ for 7min. The amplified product was detected by 1% agarose gel electrophoresis and recovered using the PCR product recovery kit SanPrep Column DNA Gel Extraction Kit. The recovered PCR product was connected to pMD19-TVector at 16°C and then transformed into E.coli DH5α competent cells. The cells were inverted and cultured overnight. Single colonies were picked for PCR verification. The amplified product was detected by 1% agarose gel electrophoresis, and the successful connection was preliminarily determined based on the electrophoresis bands. The bacterial solution containing the target bands was sent to Shanghai Bioengineering Co., Ltd. for sequencing.
[0032] 1.4 The degradation method involved in the embodiment is as follows: (1) Degradation of PET film: Bacterial suspension of target strain YL3 (OD 600 =1.0) was inoculated into 30 mL of an inorganic salt liquid culture medium containing 1 g / L PET film at an inoculum size of 3 vol%, and three parallel groups were set up (the control group was a culture medium without bacterial solution added). After culturing at 30 ° C and 180 rpm in the dark for 3 d, 7 d, and 10 d, samples were taken for HPLC determination to analyze the degradation of PET film by strain YL3.
[0033] 1.4 The sample processing and detection methods involved in the embodiment are as follows: (1) Sample treatment: 10 mL of culture medium was centrifuged at 4°C and 5000 g for 10 min, and the supernatant was extracted with 10 mL of ethyl acetate in a volume ratio of 1:1. The organic phase was rotary evaporated, and then 2 mL of chromatographic grade methanol was added for dissolution. The solution was filtered through a 0.22 μm organic membrane and then tested by HPLC.
[0034] (2) HPLC detection conditions: Chromatographic column: Shimadzu C18 column (4.6*150 mm; 5 μm); column temperature: 35°C; flow rate: 0.5 mL / min; detection wavelength: 240 nm; injection volume: 5 μL; mobile phase: 50% chromatographic grade methanol; running time: 25 min.
[0035] (3) PET film weight loss rate detection Weight loss rate of PET film (%) = [(m2-m1)÷m2]×100; m1: The PET film treated with the strain was repeatedly washed with deionized water for 3 to 5 times, placed in a sterile centrifuge tube containing 30 mL of 2% SDS solution, shaken at 37°C and 180 rpm for 2 hours, placed in an ultrasonic cleaner for 30 minutes, then washed several times with 70% anhydrous ethanol, and finally placed in a 100°C oven for drying and weighed; m2: The PET film before strain treatment was repeatedly washed with deionized water for 3 to 5 times, placed in a sterile centrifuge tube containing 30 mL of 2% SDS solution, shaken at 37°C and 180 rpm for 2 hours, placed in an ultrasonic cleaner for 30 minutes, then washed several times with 70% anhydrous ethanol, and finally placed in a 100°C oven for drying and weighed; (4) SEM analysis of PET film The PET film treated with the strain was washed with deionized water for 3 to 5 times, placed in a sterile centrifuge tube containing 30 mL of 2% SDS solution, oscillated at 37°C and 230 rpm for 2 hours, placed in an ultrasonic cleaner for 30 minutes, then washed several times with 70% anhydrous ethanol, and finally placed in a 100°C oven for drying. The PET film not treated with the strain was used as a control. A conductive tape was applied to the sample stage, and the PET film sample was picked up with tweezers and attached to the conductive tape. The sample stage with the PET film sample was placed in an ion sputtering instrument. After gold was sprayed under vacuum for 60 seconds, it was placed under a scanning electron microscope (SEM) to adjust the magnification and focus to observe its microscopic morphology. After confirmation, the image was scanned, photographed and saved.
[0036] (5) FTIR detection of PET film The PET film treated with the strain was washed with deionized water for 3 to 5 times, placed in a sterile centrifuge tube containing 30 mL of 2% SDS solution, shaken at 37°C and 230 rpm for 2 hours, placed in an ultrasonic cleaner for 30 minutes, then washed several times with 70% anhydrous ethanol, and finally dried in an oven at 100°C. The PET film not treated with the strain was used as a control. FTIR was used to characterize the changes in the functional groups on the PET surface, and the ATR mode was used to obtain the 400-4000cm -1 The spectrum was scanned 32 times on average.
[0037] (6) Degradation of PET intermediate degradation products by strain YL3 Take out the strain YL3 preserved with 50 vol% glycerol from the -80℃ freezer, put it in 4℃ to thaw for 30 min, then take 200 μL of bacterial solution in a clean bench and add it to a 10 mL LB liquid culture medium test tube, shake it at 30℃ and 180 rpm for 12-16 h to make its OD 600 When the inoculum reached 1.0, 3% (0.6 mL) of the inoculum was added to the MSM liquid medium containing 100 mg / L of BHET, MHET or TPA, and 3 parallel groups were set up for each group. Samples were taken to measure OD at 6 h, 24 h, 48 h, 3 d, 5 d and 7 d of degradation. 600 The values and the residual amounts of BHET, MHET and TPA were detected by HPLC.
[0038] 2 Results and analysis 2.1 Colony morphology of PET-degrading bacteria YL3 Pick a loop of strain YL3 and inoculate it into LB solid medium for streak culture. After culturing at 30℃ for 24h, observe its colonies. Figure 1 As shown, the single bacteria are round, light yellow, with a protrusion in the middle, neat edges and a shiny colony surface.
[0039] 2.2 Molecular biological identification of PET-degrading bacteria YL3 The 16Sr RNA gene sequence of strain YL3 is shown in SEQ ID NO. 1. The sequence was analyzed and compared in the EzBiocloud database, and the sequence was highly related to Pseudomonas plecoglossicida bacteria, and the sequence with the highest homology was the sequence of Pseudomonas plecoglossicida NBRC 103162, with a similarity of 99.93%.
[0040] Based on the above identification results, strain YL3 was identified as Pseudomonas plecoglossicida. Pseudomonas plecoglossicida YL3 was deposited in the General Microbiology Center of China Microbiological Culture Collection Administration on September 9, 2024. The deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC NO.31905.
[0041] 2.3 HPLC analysis of PET degradation by PET-degrading bacteria YL3 Figure 3This is the HPLC graph of PET film degradation by Pseudomonas proteus YL3. Compared with the blank control group, many new product absorption peaks appeared in the culture medium after 3d, 7d, and 10d of treatment with the strain, indicating that Pseudomonas proteus YL3 has degradation activity on PET film.
[0042] After being treated with Pseudomonas proteus YL3 at 30°C for 11 days, the weight of the PET film was reduced from the original 0.03g to 0.0289g, the weight loss in 11 days was 1.1mg, and the weight loss rate of the PET film reached 3.67%, which further illustrates the degradation effect of this strain on the PET film. According to literature reports, the weight loss rate of PET film after 40 days of treatment with the single bacterium B. gottheilii isolated from mangrove sediments was 3%; after 8 weeks of cultivation at 30°C and pH 7.0, the mass of PET film was reduced by about 30%, and the utilization rate was about 0.03 mg / d; after 28 days of reaction at 30°C by strain Penicillium simplicissimum 28f2, the weight loss rate of PC-PET film reached 3.09%; after 4 weeks of cultivation at 30°C and pH 7.0 by Pseudomonas sp. TDA1, the mass of PET film was reduced by about 20%, and its degradation rate was about 0.05 mg / d.
[0043] 2.4 SEM analysis of PET degradation by PET-degrading bacteria YL3 like Figure 4 As shown in the figure, by taking out the PET film from the culture medium and directly performing gold spray SEM without washing, we can see that Pseudomonas proteans YL3 colonizes on the film surface and uses PET to produce PET fragments, which shows that the process of Pseudomonas proteans YL3 using PET starts with colonization. Figure 5 It can be seen that the PET film treated with Pseudomonas proteans YL3 and the PET film not treated with the strain were magnified 300 times, 600 times, and 2500 times. It can be seen that the surface of the PET film not treated with Pseudomonas proteans YL3 is flat and smooth, but the surface of the PET film treated with Pseudomonas proteans YL3 is rough and has obvious cracks, and PET fragments can be observed. The generation of PET film surface fragments and the process from colonization to degradation of Pseudomonas proteans YL3 on PET film further prove that Pseudomonas proteans YL3 has a high degradation ability on PET film.
[0044] 2.5 FTIR analysis of PET degradation by PET-degrading bacteria YL3 PET is a polyester compound, and the ester bond is the most critical functional group. FTIR results are as follows Figure 6As shown in the figure, compared with the PET film not treated with Pseudomonas proteus YL3, the PET film treated with Pseudomonas proteus YL3 had a -1 CH bond, 1000-1300cm -1 CO bond between 1700-1730cm -1 The key functional groups such as the C=O bonds between PET are reduced. They break the ester bonds of PET and convert large molecular insoluble substances into small molecular soluble substances, thereby achieving the purpose of degrading PET.
[0045] 2.6 Degradation of PET intermediate degradation products BHET, MHET, and TPA by PET-degrading bacteria YL3 The degradation of PET intermediate degradation products BHET, MHET, and TPA by Pseudomonas proteus YL3 was analyzed. Figure 7 As shown in Figure 2, the growth rate and OD of Pseudomonas proteus YL3 in MSM liquid medium containing BHET and MHET during the whole culture process. 600 The values were similar. In the first 48 h, the degradation rates and OD values of the three PET intermediate degradation products of the strain Pseudomonas proteus YL3 were 600 The values showed an overall increasing trend with the extension of time, especially at the 24th hour, when the two PET intermediate degradation products were rapidly degraded. 600 The value also rises rapidly. At this time, the OD 600 The values were 0.89 and 0.71, respectively. At 48h, the degradation rates of the intermediate degradation products had reached more than 95%. Pseudomonas proteus YL3 began to detect the degradation of TPA at 48h, and then further degradation of TPA could be observed in the subsequent daily tests. The degradation rate reached 68% at 120h and 95% at 168h. 600 The increase in the expression of Pseudomonas aeruginosa YL3 indicated that the strain was growing continuously. In summary, Pseudomonas aeruginosa YL3 could effectively degrade the intermediate degradation products of PET, including BHET, MHET and TPA, which further proved that Pseudomonas aeruginosa YL3 could effectively degrade PET.
[0046] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention should fall within the scope of the present invention.
Claims
1. A strain of Pseudomonas proteus, characterized in that: The Pseudomonas proteus is named as Pseudomonas proteus ( pseudomonas plecoglossicida )YL3, deposited on September 9, 2024 in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit address at No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC NO.31905.
2. Use of the Pseudomonas proteus according to claim 1 in degrading polyethylene terephthalate and its intermediate degradation products.
3. The use according to claim 2, characterized in that: The polyethylene terephthalate intermediate degradation products include dihydroxyethyl terephthalate, ethylene terephthalate and terephthalic acid.
4. Use of the Pseudomonas proteus according to claim 1 in preparing a bacterial agent for degrading polyethylene terephthalate and its intermediate degradation products.
5. A bacterial agent, characterized in that: The bacterial agent contains the Pseudomonas aeruginosa according to claim 1.
6. Use of the bacterial agent as claimed in claim 5 in degrading polyethylene terephthalate and its intermediate degradation products.
7. The use according to claim 6, characterized in that: The polyethylene terephthalate intermediate degradation products include dihydroxyethyl terephthalate, ethylene terephthalate and terephthalic acid.
8. A method for degrading polyethylene terephthalate, characterized in that: The Pseudomonas proteus according to claim 1 or the bacterial agent according to claim 5 is contacted with polyethylene terephthalate and degraded at 30°C.
9. A method for degrading intermediate degradation products of polyethylene terephthalate, characterized in that: The Pseudomonas proteus described in claim 1 or the bacterial agent described in claim 5 is contacted with the intermediate degradation product of polyethylene terephthalate and degraded at 30°C.
10. The method according to claim 9, characterized in that: The polyethylene terephthalate intermediate degradation products include dihydroxyethyl terephthalate, ethylene terephthalate and terephthalic acid.