Acinetobacter W107 and application thereof in degradation of polyethylene glycol terephthalate microplastics
By screening and identifying Acinetobacter soli W107 strains of Acinetobacter soli, the problem of low degradation efficiency of PET microplastics in the prior art was solved, and the significant degradation effect of PET microplastics was achieved, providing an economical and environmentally friendly biodegradation method.
Patent Information
- Application Number
- CN202411887730.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-20
- Publication Date
- 2025-04-18
- Estimated Expiration
- Not applicable · inactive patent
AI Technical Summary
Existing microorganisms have low degradation efficiency of polyethylene terephthalate (PET) microplastics and have harsh degradation conditions, making it difficult to develop efficient biodegradation methods.
The Acinetobacter soli W107 strain of Acinetobacter genus was screened and identified, which was able to be easily cultivated and reproduced on artificial culture medium and showed significant PET microplastic degradation ability.
The W107 strain showed a degradation efficiency of 11.4% in degrading PET microplastics, effectively treating PET microplastics through enzymatic and biodegradable mechanisms to reduce environmental pollution.
Smart Images
Figure CN119842521A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of microbial technology, and in particular to Acinetobacter W107 and application thereof in degrading polyethylene terephthalate microplastics. Background Art
[0002] As one of the most influential inventions of the 20th century, plastics play a vital role in many industries such as packaging, construction, and agriculture. Although plastics have greatly facilitated human life, they also pose a severe challenge to the natural environment. Among the many types of plastics, PET plastics occupy a prominent position. According to statistics, about 60% of plastic waste is PET products. Microplastics, that is, plastic particles with a diameter of less than 5 mm, including nano-scale particles, have caused increasingly serious pollution problems in soil, oceans, freshwater and other environments. Therefore, there is an urgent need for an economical and environmentally friendly method to efficiently recycle polyester plastic waste.
[0003] Plastic recycling methods are mainly divided into physical recycling, chemical recycling and biological recycling. Physical recycling and chemical recycling will cause a certain degree of damage to plastics when processing them and may cause environmental pollution. As a sustainable recycling method, biological recycling is centered on biodegradation, such as using microorganisms and enzymes to decompose plastics into environmentally friendly oligomers or monomers.
[0004] At present, some microorganisms have been found to be able to degrade polyethylene terephthalate (PET) plastics, but the degradation efficiency of these microorganisms is usually low and the degradation conditions are relatively harsh. Therefore, developing a microbial strain that can efficiently degrade PET microplastics has important practical application value. Summary of the invention
[0005] In view of this, the present invention proposes a Bacterium W107 and its application in the degradation of polyethylene terephthalate microplastics, aiming to screen new strains, explore their degradation mechanisms, establish an efficient PET degradation system, develop an economical and environmentally friendly degradation method, and provide more theoretical basis and practical evidence for PET degradation research.
[0006] The invention provides an acinetobacter, which is an acinetobacter of the genus Acinetobacter soliW107, and the preservation number of the acinetobacter in the China Center for Type Culture Collection is CCTCC NO: M20242613.
[0007] The present invention also provides a bacterial agent, which contains the Acinetobacter.
[0008] The present invention also provides a fermentation broth, which is obtained by fermenting the Acinetobacter.
[0009] The present invention also provides a composition, which comprises the Acinetobacter and / or the bacterial agent and / or the fermentation broth.
[0010] The present invention also proposes an application of the Acinetobacter in any of the following:
[0011] A1) Application in the production of lipase and / or application in the preparation of lipase products;
[0012] A2) use in the production of esterase and / or use in the preparation of esterase products;
[0013] A3) Application in the degradation of PET and / or application in the preparation of degraded PET products.
[0014] The present invention also proposes an application of the bacterial agent in any of the following:
[0015] A1) Application in the production of lipase and / or application in the preparation of lipase products;
[0016] A2) use in the production of esterase and / or use in the preparation of esterase products;
[0017] A3) Application in the degradation of PET and / or application in the preparation of degraded PET products.
[0018] The present invention also proposes an application of the composition in any of the following:
[0019] A1) Application in the production of lipase and / or application in the preparation of lipase products;
[0020] A2) use in the production of esterase and / or use in the preparation of esterase products;
[0021] A3) Application in the degradation of PET and / or application in the preparation of degraded PET products.
[0022] The present invention also provides a method for preparing an enzymatic hydrolysis product, the method comprising the steps of reacting a fat substrate with the Acinetobacter;
[0023] Preferably, the fat substrate is p-nitrophenol palmitate.
[0024] The present invention also provides a method for preparing an enzymatic hydrolysis product, the method comprising the steps of reacting an ester substrate with the Acinetobacter;
[0025] Preferably, the ester substrate is p-nitrophenyl acetate.
[0026] The present invention also provides a method for preparing a degradation product, which comprises the step of reacting PET with the Acinetobacter.
[0027] Compared with the prior art, the present invention has the following beneficial effects:
[0028] The Acinetobacter soli W107 strain involved in the present invention can be successfully cultured on an artificial culture medium and is easy to obtain and reproduce, which is crucial for being a strain for degrading microplastics and facilitates its application and popularization in industrial production. The W107 strain exhibits significant degradation ability for PET microplastics, with a degradation efficiency of up to 11.4%.
[0029] The present invention uses microbial degradation technology to effectively treat PET microplastics, thereby reducing the impact on the environment. Compared with other degradation technologies, this method avoids the environmental pollution problems that may be caused. BRIEF DESCRIPTION OF THE DRAWINGS
[0030] Various other advantages and benefits will become apparent to those of ordinary skill in the art by reading the detailed description of the preferred embodiments below. The accompanying drawings are only for the purpose of illustrating the preferred embodiments and are not to be considered as limiting the present invention. Moreover, the same reference symbols are used throughout the accompanying drawings to represent the same components. In the accompanying drawings:
[0031] Figure 1 This is a plate colony image of Acinetobacter soliW107 cultured at 37°C;
[0032] Figure 2 The results of the lipase and esterase tests are shown in Figure 1, where A is the result of the esterase activity test and B is the result of the lipase activity test;
[0033] Figure 3 The electron microscope results of the PET particle degradation test are shown in FIG. 1 , wherein (a) and (b) are scanning electron microscope images of the PET particles before being treated with Acinetobacter soli W107 at 200 times and 500 times magnification, respectively, and (c) and (d) are scanning electron microscope images of the PET particles after being treated at 200 times and 500 times magnification, respectively;
[0034] Figure 4 The figures are high performance liquid chromatograms of PET particle degradation products, wherein (a) is a high performance liquid chromatogram of 0.005 mM TPA standard, and (b) is a high performance liquid chromatogram of PET particle degradation products in the reaction solution after treatment with Acinetobacter soliW107 for 15 days. DETAILED DESCRIPTION
[0035] Exemplary embodiments of the present disclosure will be described in more detail below with reference to the accompanying drawings. Although exemplary embodiments of the present disclosure are shown in the accompanying drawings, it should be understood that the present disclosure can be implemented in various forms and should not be limited by the embodiments described herein. On the contrary, these embodiments are provided in order to enable a more thorough understanding of the present disclosure and to fully convey the scope of the present disclosure to those skilled in the art. It should be noted that, in the absence of conflict, the embodiments of the present invention and the features described in the embodiments can be combined with each other. The present invention will be described in detail below with reference to the accompanying drawings and in combination with the embodiments.
[0036] Example 1 Isolation of Acinetobacter soli W107
[0037] The strain provided by the present invention is Acinetobacter soli W107, which is screened from samples collected from abandoned ditches around Nanyang Jinda Oil Transportation Co., Ltd. that are seriously polluted by plastics. The isolation process is as follows:
[0038] Take 5 grams of samples collected from abandoned ditches with serious plastic pollution around Nanyang Jinda Oil Transportation Co., Ltd., add them to a triangular flask containing 45 ml of sterile water, and culture them in a shaker at 28°C and 180 rpm for 24 hours. After standing for 30 minutes, take 100 microliters of supernatant in the clean bench for dilution and spread on LB solid culture medium. The coated culture medium was placed in a constant temperature incubator at 37°C for 12 hours, and then different single colonies were selected for transfer using an inoculation loop and placed on an LB solid plate for purification for subsequent biological activity testing. Through this series of operations, we screened strain W107, Acinetobacter soliW107.
[0039] Acinetobacter soli W107 exhibits the following biological characteristics: it is round, translucent, has neat edges, and a smooth and moist surface on LB plate culture medium. Through 16S rRNA gene sequencing and nucleic acid sequence homology comparison with similar sequences already in the GenBank database, it was found that W107 has the highest similarity with Acinetobactersoli ON778563.1, reaching 99.72%. Acinetobacter soli W107 was deposited in the China Center for Type Culture Collection (address: Wuhan University, No. 229, Bayi Road, Wuchang District, Wuhan City, Hubei Province) on November 21, 2024, with a deposit number of CCTCC NO: M 20242613.
[0040] This strain can grow with PET particles as the only carbon source and has the ability to degrade PET microplastics.
[0041] Example 2
[0042] 1. Cultivation and fermentation process of Acinetobacter soli W107
[0043] The W107 bacteria were inoculated into LB liquid culture tubes (5 ml per tube), and cultured at 37°C and 200 rpm in a shaking incubator for 12 h to obtain test tube cultures.
[0044] Transfer the test tube culture into a fresh LB liquid culture medium test tube (5 ml per tube), and culture at 37°C and 200 rpm in a shaking incubator for 12 hours to obtain a bacterial solution with strong activity.
[0045] Take 100 μL of the above bacterial solution and spread it on fresh LB solid culture medium, and culture it in a 37°C incubator for 12 h to obtain plate bacteria. Figure 1 This is a plate colony image of Acinetobacter soli W107 cultured at 37°C.
[0046] 2. Lipase and esterase production test of Acinetobacter soli W107
[0047] 1. Determination of lipase and esterase activities of Acinetobacter soli W107 using the p-nitrophenol (p-NP) method
[0048] (1) Determination of lipase activity: p-nitrophenol palmitate (p-NPP) was used as a substrate, and the lipase activity was determined according to the p-nitrophenol (p-NP) method, and a standard curve of p-nitrophenol concentration was drawn. The specific operation steps are as follows:
[0049] Drawing of the p-nitrophenol concentration standard curve: Weigh 0.08346 g of p-NP (6 mmol), dissolve it with an appropriate amount of 95% ethanol, and dilute it to 100 ml with distilled water. Take 0, 2.5, 5, 7.5, 10, 15, 20, 30, 40, and 50 μl of p-NP standard solution and add it to a test tube, then add 50 mmol / L Tris-HCl buffer (pH 8.0) to dilute it to 2 ml. Add 1 ml of 95% ethanol solution to the test tubes containing different concentrations of p-NP, mix thoroughly, and measure the absorbance at 410 nm to draw the standard curve of p-nitrophenol solution concentration.
[0050] Preparation of the mixed solution: 10 mmol / L p-nitrophenol palmitate and 50 mmol / L Tris-HCl buffer (pH 8.0) were mixed at a ratio of 1:9, and 1% Triton X-100 and 0.1% gum arabic powder were added.
[0051] Enzyme activity determination: Take 0.1 ml of enzyme solution (diluted 1 times) and add 0.9 ml of mixed solution. The reaction conditions are set to 30°C and water bath for 10 minutes. Add 1 ml of 95% ethanol to terminate the reaction and measure the absorbance at 410 nm. Use the enzyme solution inactivated in a 95°C water bath as a control and calculate the lipase activity based on the p-nitrophenol concentration standard curve.
[0052] (2) Determination of esterase activity
[0053] Using p-nitrophenyl acetate (PNPA) as a substrate, the esterase activity was determined according to the p-nitrophenol (p-NP) method, and a standard curve of p-nitrophenol concentration was drawn. The specific steps are as follows:
[0054] Drawing of the p-nitrophenol concentration standard curve: Weigh 0.08346 g of p-NP (6 mmol), dissolve it with an appropriate amount of 95% ethanol, and dilute it to 100 ml with distilled water. Take 0, 2.5, 5, 7.5, 10, 15, 20, 30, 40, and 50 μl of p-NP standard solution and add it to a test tube, then add 50 mmol / L phosphate buffer (pH 6.5) to dilute it to 2 ml. Add 1 ml of 95% ethanol solution to the test tubes containing different concentrations of p-NP, mix thoroughly, and measure the absorbance at 405 nm to draw the standard curve of p-nitrophenol solution concentration.
[0055] Preparation of the mixed solution: Mix 1 mmol / L p-nitrophenyl acetate and phosphate buffer (pH 6.5) in a ratio of 1:9.
[0056] Enzyme activity determination: Take 0.1 ml of enzyme solution and add 0.9 ml of mixed solution. The reaction conditions are set to 30°C and water bath for 10 minutes. Add 1 ml of 0.5 mol / L NaOH to terminate the reaction and measure the absorbance at 405 nm. Use the reacted mixed solution as a control and calculate the esterase activity based on the p-nitrophenol concentration standard curve.
[0057] 2. 30-day shake flask test with PET particles as the only carbon source
[0058] (1) Pretreatment and sterilization of PET particles: First, the purchased PET particles were soaked in 75% anhydrous ethanol for 36 hours, then taken out and transferred to 20% anhydrous ethanol, and ultrasonically cleaned for 30 minutes using an ultrasonic cleaner, and repeated 3-5 times. Then, they were washed 3 times with sterile deionized water and irradiated with ultraviolet light for 48 hours in a clean bench to ensure adequate sterilization.
[0059] The Acinetobacter soli W107 strain was inoculated into a sterilized LB liquid medium for culture. After the strain grew to the logarithmic phase, the culture was centrifuged in a centrifuge at 8000r / min for 10 minutes. After centrifugation, the supernatant was poured out, sterilized saline was added and centrifuged again in a centrifuge at 8000r / min for 10 minutes, the supernatant was discarded, and repeated twice. Physiological saline was added for the last time to disperse the bacteria, and the above operations were all performed in a clean bench. Take 2mL of each activated strain and inoculate it in 30mL of inorganic salt medium containing 2g of PET microplastics. The inorganic salt medium containing PET microplastics but not inoculated with W107 bacteria was used as the control group, and was shaken and cultured at 30°C and 180r / min for 30 days, with 3 parallel experiments set up for each group. After 30 days, the weight loss of PET particles was measured to investigate its biodegradation characteristics.
[0060] (2) Calculation of microplastic weight loss rate
[0061] PET microplastic particles were removed from the culture medium, washed with 75% ethanol, and then dried at 60°C overnight to remove the biofilm attached to the plastic particles. The weight of the residual polymer was measured using an analytical balance with an accuracy of 0.0001g to assess the extent of biodegradation.
[0062] The weight loss rate of microplastics is calculated according to the following formula:
[0063] Weight loss rate of microplastics = (initial weight - final weight) / initial weight × 100%.
[0064] (3) Scanning electron microscopy
[0065] 3. High performance liquid chromatography (HPLC) analysis and detection
[0066] (1) Sample preparation
[0067] The standard TPA was dissolved in dimethyl sulfoxide (DMSO) to prepare a 0.005 mM standard solution; the sample supernatant and the standard solution were passed through a 0.22 μM filter membrane and then subjected to HPLC analysis.
[0068] (2) Test conditions
[0069] Using C 18The reverse phase column was used as the chromatographic column; the column temperature was set to 25°C; the detection wavelength was set to 254 nm; and the flow rate was set to 1 mL min -1 ; The injection volume was 10 μL; The mobile phase was formic acid (0.1%) / water / acetonitrile (50:30:20, v / v / v). HPLC detection was performed on the reaction solution of the Acinetobacter soli W107 strain on the 15th day of the shake flask test.
[0070] 4. Test results
[0071] (1) Results of lipase and esterase production tests are shown in the figure below Figure 2 As shown, A is the result of esterase activity detection, and B is the result of lipase activity detection. The enzyme activity was quantitatively calculated by the p-nitrophenol (p-NP) method. The lipase activity was 0.077U / ml, and the esterase activity was 0.076U / ml.
[0072] (2) The degradation rate of Acinetobacter soliW107 strain was 11.4% at 30 days.
[0073] (3) Figure 3 The electron microscope results of the PET particle degradation test are shown in Figure 1, where (a) and (b) are scanning electron microscope images of PET particles before being treated with Acinetobacter soliW107 at 200 times and 500 times magnification, respectively, and (c) and (d) are scanning electron microscope images of PET particles after treatment at 200 times and 500 times magnification, respectively. Figure 3 It can be seen that obvious erosion and holes appeared on the surface of PET particles, indicating that the W107 strain has a significant degradation effect on PET particles.
[0074] (4) Figure 4 The HPLC results are shown in Figure 1, where (a) is a HPLC chromatogram of a 0.005 mM TPA standard product and (b) is a HPLC chromatogram of degradation products of PET particles in the reaction solution after treatment with Acinetobacter soli W107 for 15 days. Figure 4 It can be seen that terephthalic acid (TPA) was detected in the reaction solution on the 15th day, which indicated that the W107 strain produced TPA during the process of degrading PET, further confirming that the strain had the ability to degrade PET.
[0075] In summary, Acinetobacter soli W107 strain showed significant biological activity in degrading PET microplastics. Its degradation ability was not only confirmed by weight loss rate and scanning electron microscopy observation, but also by HPLC analysis to detect the degradation product terephthalic acid (TPA), thus providing new possibilities for the biodegradation of PET plastics. The research results provide an important scientific basis for the development of new biodegradable plastics and provide potential microbial resources for the bioremediation of plastic pollution.
[0076] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, ordinary technicians in the relevant field should understand that the specific implementation methods of the present invention can still be modified or replaced by equivalents. Any modification or equivalent replacement that does not depart from the spirit and scope of the present invention should be covered within the scope of protection of the claims of the present invention.
Claims
1. An Acinetobacter, characterized in that The Acinetobacter is Acinetobacter soli W107, and its deposit number in China Center for Type Culture Collection is CCTCC NO: M20242613.
2. A bacterial agent, characterized in that The bacterial agent contains the Acinetobacter described in claim 1.
3. A fermentation broth, characterized in that: The fermentation broth is obtained by fermenting the Acinetobacter described in claim 1.
4. A composition, characterized in that The composition comprises the Acinetobacter described in claim 1 and / or the bacterial agent described in claim 2 and / or the fermentation broth described in claim 3.
5. Use of the Acinetobacter according to claim 1 in any of the following: A1) Application in the production of lipase and / or application in the preparation of lipase products; A2) use in the production of esterase and / or use in the preparation of esterase products; A3) Application in the degradation of PET and / or application in the preparation of degraded PET products.
6. Use of the bacterial agent according to claim 2 in any of the following: A1) Application in the production of lipase and / or application in the preparation of lipase products; A2) use in the production of esterase and / or use in the preparation of esterase products; A3) Application in the degradation of PET and / or application in the preparation of degraded PET products.
7. Use of the composition according to claim 4 in any of the following: A1) Application in the production of lipase and / or application in the preparation of lipase products; A2) use in the production of esterase and / or use in the preparation of esterase products; A3) Application in the degradation of PET and / or application in the preparation of degraded PET products.
8. A method for preparing an enzymatic hydrolysis product, characterized in that: The method comprises the steps of reacting a fat substrate with the Acinetobacter described in claim 1; The fat substrate is p-nitrophenol palmitate.
9. A method for preparing an enzymatic hydrolysis product, characterized in that: The method comprises the steps of reacting an ester substrate with the Acinetobacter described in claim 1; The ester substrate is p-nitrophenyl acetate.
10. A method for preparing a degradation product, characterized in that: The method comprises the step of reacting PET with the Acinetobacter described in claim 1.