Geotrichum ETG with lipase and esterase activity and application of geotrichum ETG in plastic degradation

Abstract

The invention relates to the technical field of microorganisms, and discloses a geotrichum ETG with lipase and esterase activity and application of the geotrichum ETG in plastic degradation, the geotrichum ETG is geotrichum sp.ETG, and the preservation number of the geotrichum ETG in the China Center for Type Culture Collection is CCTCC NO: M 20242614. The geotrichum sp. ETG provided by the invention can be easily cultured on an artificial culture medium and is easy to obtain and breed, the geotrichum sp. ETG shows remarkable degradation capacity on PET plastic particles, and the weight loss rate of the geotrichum sp. ETG is up to 18.3%.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and in particular to Geotrichum ETG having lipase and esterase activities and application thereof in plastic degradation. Background Art

[0002] Plastic materials play an indispensable role in many applications due to their excellent stability, durability and outstanding performance. However, the main problem of plastic materials is their long-term persistence in the environment. The continuous accumulation of plastic waste in terrestrial and marine environments has become a concern, mainly due to the low natural degradation ability of plastics and the imperfect recycling mechanism.

[0003] Currently, common methods for dealing with plastic waste include mechanical recycling, landfill, chemical treatment, thermal degradation, photodegradation, advanced oxidation processes (AOPs) and biological treatment. Existing waste disposal methods are often costly, lack sustainability, and have a significant impact on the environment. Among these methods, biological treatment is considered to be the most consistent with the concept of sustainable development. Studies have shown that specific insects, bacteria and fungi can decompose plastic polymers and convert them into carbon compounds that are harmless to the environment. Therefore, biodegradation is considered to be the most promising plastic degradation pathway in the future.

[0004] At present, although some studies have reported the potential of certain microorganisms in plastic degradation, the degradation efficiency and application range of these microorganisms are still limited. Therefore, the development of new microbial resources, especially those that can efficiently degrade plastics, is of great significance to solving the problem of plastic pollution. Summary of the invention

[0005] In view of this, the present invention proposes a Geotrichum ETG having lipase and esterase activities and its application in plastic degradation, aiming to provide a new microbial resource to improve the biodegradation efficiency of plastics and expand its scope of application.

[0006] The invention provides a geotrichum sp., which is Geotrichum sp. ETG, and its preservation number in China Center for Type Culture Collection is CCTCC NO: M 20242614.

[0007] The present invention also provides a bacterial agent, which contains the Geotrichum sp.

[0008] The present invention also provides a fermentation liquid, which is obtained by fermenting the Geotrichum sp.

[0009] The present invention also provides a composition, which comprises the Geotrichum and / or the bacterial agent and / or the fermentation liquid.

[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 Geotrichum;

[0023] Preferably, the fat substrate is rhodamine B.

[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 Geotrichum;

[0025] Preferably, the ester substrate is tributyrin.

[0026] The present invention also provides a method for preparing a degradation product, which comprises the step of reacting PET with the Geotrichum sp.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] The Geotrichum sp. ETG involved in the present invention can be easily cultured on an artificial culture medium, and is easy to obtain and reproduce, which is crucial for waste treatment strains and helps to achieve industrial production and wide promotion. The fungus Geotrichum sp. ETG exhibits significant degradation ability for PET plastic particles, with a weight loss rate of up to 18.3%.

[0029] The present invention successfully degrades PET plastic particles by using biotechnology, and effectively avoids problems such as environmental pollution compared with chemical agents and physical methods. 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 It is one of the results of the ETG lipase plate test of Geotri chum sp.;

[0032] Figure 2 This is the second result of the Geotri chum sp. ETG lipase plate test;

[0033] Figure 3 The scanning electron microscope images of plastic particles before and after the degradation experiment, where A is before the experiment and B is after the experiment;

[0034] Figure 4 The results of HPLC test, where A is 0.005 mM TPA and B is the fermentation liquid of PET treated with ETG bacteria for 30 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] The culture medium formula used in the present invention is as follows:

[0037] The formula of YPD solid medium includes: 2% glucose; 1% yeast extract; 2% peptone; 2% agar; and deionized water.

[0038] The formula of YPD liquid medium includes: 2% glucose; 1% yeast extract; 2% peptone; and deionized water.

[0039] The formula (g / L) of the Rhodamine B (lipase) solid culture medium is: 1) 9.0 nutrient broth powder, 2.5 yeast extract, 18-20 agar, 950 ml water, and autoclaved at 121°C for 20 minutes; 2) Rhodamine B with a final concentration of 0.0012% (wt / vol), 250 μl Tween 80, 30 ml olive oil, and 20 ml double distilled water are mixed to form an emulsion, which is added to 1) which has been sterilized and cooled to 45°C.

[0040] The formula of 1% tributyrin (esterase) solid medium is: 1% tributyrin, 2% glucose; 1% yeast extract; 2% peptone; 2% agar; and deionized water.

[0041] The formula (g / L) of the inorganic salt solid culture medium with PET as the sole carbon source is: 1) 1.0 potassium dihydrogen phosphate, 1.5 disodium hydrogen phosphate, 2.0 ammonium chloride, 0.1 calcium chloride dihydrate, 0.15 potassium chloride, 0.2 magnesium sulfate heptahydrate, 0.01 zinc sulfate heptahydrate, 0.001 manganese sulfate monohydrate, 18 agar, 121°C, high pressure sterilization for 20 minutes; 2) 0.03 PET powder was placed on a clean bench under ultraviolet light for 3 hours and then added to 1) at 45°C.

[0042] The formula (g / L) of the inorganic salt liquid culture medium is: 1) 1.0 potassium dihydrogen phosphate, 1.5 disodium hydrogen phosphate, 2.0 ammonium chloride, 0.1 calcium chloride dihydrate, 0.15 potassium chloride, 0.2 magnesium sulfate heptahydrate, 0.01 zinc sulfate heptahydrate, and 0.001 manganese sulfate monohydrate.

[0043] The formula of the seed liquid culture medium includes: 2% glucose; 1% yeast extract; 2% peptone; and deionized water.

[0044] Example 1 Isolation of Geotrichum sp. ETG

[0045] The strain involved in the present invention is Geotrichum sp.ETG, which is derived from water samples of rivers around an oil transportation company in Nanyang City, Henan Province.

[0046] The isolation steps of ETG strains are as follows:

[0047] 1) Put 400 ml of river water sample into 500 ml blue-capped bottle and bring it back to the laboratory; 2) Use ultrafiltration device to filter the collected water sample, using a 0.22 micron water filter membrane for every 100 ml of water sample; 3) Take a piece of filtered filter membrane, add it to 100 ml of MSM liquid culture medium containing 0.3 g of PET powder and 250 μl of 100 mg / ml chloramphenicol, and culture it at 30°C and 180 rpm for 20 days; 4) Culture 2 0 days later, take 100 microliters of the pre-cultured suspension and spread it on a YPD solid medium containing chloramphenicol; 5) Place the coated medium in a 30°C biochemical incubator for 2-3 days, and after the single colony grows to an appropriate size, select yeast colonies of different colors and shapes for numbering and recording; 6) Use an inoculation loop to select single colonies that are obvious and have different shapes on the culture medium for secondary or multiple purifications, and transfer them to a new YPD solid medium for subsequent identification and biological activity testing. Through the above steps, the strain ETG of the present invention, Geotrichum sp. ETG, was successfully isolated.

[0048] The strain ETG has the following biological characteristics: the colonies on the YPD plate medium are white, dry, with protrusions in the center and hairy edges. The results of LSU RNA gene sequence determination and nucleic acid sequence homology comparison with other similar sequences included in the GenBank database showed that ETG had the highest similarity with Geotrichus sp. strain 186CK-3-2 (99.45%). The Geotrichus sp. ETG 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 the collection number: CCTCC NO: M20242614. The strain has the function of degrading PET plastic particles.

[0049] Example 2

[0050] 1. Cultivation and fermentation process of Geotrichum sp.ETG

[0051] The purified Geotrichum sp. ETG bacterial cells were streaked onto YPD solid culture medium and cultured in a 30° C. biochemical incubator for 3 days to obtain plate strains.

[0052] The Geotrichum sp.ETG bacterial cells were placed in a 1 ml centrifuge tube containing 500 μl of double distilled water to prepare a Geotrichum sp.ETG bacterial suspension.

[0053] The Geotrichum sp. ETG bacterial cells were inoculated into YPD liquid culture medium (250 ml conical flask) (50 ml per flask), and cultured at 30° C. and 180 rpm shaking for 3 days to obtain Geotrichum sp. ETG seed liquid.

[0054] 2. Enzyme activity test

[0055] Take 10 μl of the above-mentioned Geotrichum suspension, spot it on the lipase and esterase activity detection plates respectively, place it in a 30°C biochemical incubator for 3-5 days, and detect whether it has the corresponding enzyme activity.

[0056] 3. PET particle degradation experiment

[0057] 1. PET pretreatment

[0058] The PET particles were weighed and immersed in 75% anhydrous ethanol for 36 hours, then taken out, placed in 20% anhydrous ethanol, and ultrasonically washed in an ultrasonic cleaner for 30 minutes, repeated 3 times, and then washed 3 times with double distilled water, and placed in a clean bench for ultraviolet irradiation for 48 hours for full sterilization.

[0059] 2. Acquisition of Geotrichum

[0060] The seed liquid of Geotrichum was collected, centrifuged at 10,000 rpm for 10 minutes, and washed three times with double distilled water to obtain the mycelium of Geotrichum.

[0061] 3. Hierarchical screening of PET plastic degradation strains

[0062] (1) Lipase plate test

[0063] Rhodamine B (lipase) solid culture medium formula (g / L): 9.0 nutrient broth powder, 2.5 yeast extract, and 18-20 g agar are dissolved in 950 ml water, and after high-pressure sterilization at 121°C for 20 minutes, an emulsion prepared by mixing Rhodamine B with a final concentration of 0.0012% (wt / vol), 250 μl Tween 80, 30 ml olive oil and 20 ml double distilled water is added, and the mixture is then added to the above culture medium that has been cooled to 45°C.

[0064] Preparation of yeast suspension: Select a single colony of isolated and purified yeast and place it in a 1 ml centrifuge tube containing 500 μl of double distilled water to prepare a yeast suspension.

[0065] Lipase activity detection: Take 10 μl of yeast suspension, apply it on the Rhodamine B solid culture medium, and then place it in a biochemical incubator at 30°C for 3-5 days. After the colonies grow, observe whether there is a red change, and check whether there is fluorescence under a 365nm ultraviolet lamp.

[0066] (2) Esterase plate test

[0067] 1% tributyrin (esterase) solid culture medium formula: 1% tributyrin, 2% glucose, 1% yeast extract, 2% peptone, 2% agar and appropriate amount of deionized water are mixed evenly.

[0068] Preparation of yeast suspension

[0069] A single colony of isolated and purified yeast was picked and placed in a 1 ml centrifuge tube containing 500 μl of double distilled water to prepare a yeast suspension.

[0070] Esterase activity detection: Take 10 μl of yeast suspension, apply it on 1% tributyrin solid culture medium, and culture it in a 30°C biochemical incubator for 3-5 days. Observe whether a transparent circle is formed around the colony.

[0071] (3) Implementation of PET particle degradation experiment

[0072] Take 0.5 grams of fungus cells and transfer them to inorganic salt liquid culture medium, add 1 PET plastic particle, and culture in a constant temperature oscillator at 30°C and 180r / min for 30 days. Microplastics were recovered from the culture medium by filtration and sieving, and ultrasonically cleaned with 75% ethanol, 2% SDS, and double distilled water in turn, and 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 0.0001 grams to evaluate the degree of biodegradation. The weight loss rate of microplastics is calculated as follows: (initial weight-weight) / initial weight × 100%.

[0073] High performance liquid chromatography (HPLC) analysis of degradation products in the culture medium after degradation experiment:

[0074] Chromatographic column: C18 reverse phase column 250mm×4.6mm, 5μm;

[0075] Mobile phase: formic acid (0.1%) / water / acetonitrile (50:30:20, v / v / v);

[0076] Flow rate: 0.8-1.0mL / min;

[0077] Detector: Use UV-Vis Detector. TPA has maximum absorption at 240-270nm, so 245nm is selected as the detection wavelength;

[0078] Column temperature: 25°C;

[0079] Injection volume: Take 20-50 μL of sample and inject it into the HPLC system;

[0080] Running time: 10-15 minutes.

[0081] IV. Results

[0082] Table 1 shows the test results of various indicators of Geotrichum sp.ETG.

[0083] Table 1

[0084]

[0085]

[0086] From Table 1, Figure 1 and Figure 2 It can be seen that Geotrichum sp. ETG has weak lipase activity and strong esterase activity. During the 30-day culture period, the degradation rate of PET plastic particles by this strain reached 18.306%. This shows that this strain has certain potential in degrading PET plastics, especially with the participation of esterase activity, it can effectively promote the biodegradation process of PET plastics.

[0087] In order to further verify the degradation ability of Geotrichum sp.ETG, the changes in the surface morphology of PET particles before and after degradation were observed by scanning electron microscopy (SEM). These analyses can provide more intuitive evidence to evaluate the degradation effect of the strain on PET plastics. The results are as follows: Figure 3 As shown. Figure 3 It can be seen that after 30 days of cultivation, the surface of the PET plastic particles treated with Geotrichum sp.ETG showed obvious erosion and depression. Compared with the untreated PET particles, the surface of the treated particles became rough with obvious cracks and holes, which shows that the Geotrichum sp.ETG can decompose the surface structure of PET plastic.

[0088] Figure 4 The figure shows the presence of terephthalic acid, a degradation product, in the fermentation broth detected by high performance liquid chromatography (HPLC) after 30 days of degradation experiment. Figure A shows a 0.005 mM terephthalic acid (TPA) standard solution, while Figure B shows the fermentation broth of PET plastic after 30 days of ETG bacterial treatment. Figure 4 It can be seen that in Figure B, a peak similar to that of the standard solution in Figure A can be observed, indicating that terephthalic acid does exist in the fermentation broth. This result further confirms that Geotrichum sp. ETG can produce terephthalic acid during the degradation of PET plastics, which is one of the main degradation products of PET plastics. The detection results of terephthalic acid provide chemical evidence for the potential of Geotrichum sp. ETG in the biodegradation of PET plastics.

[0089] In summary, Geotrichum sp.ETG shows certain application prospects in the degradation of PET plastics.

[0090] 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. A Geotrichum sp., characterized in that: The Geotrichum sp. is Geotri chum sp. ETG, and its collection number in China Center for Type Culture Collection is CCTCC NO: M 20242614.

2. A bacterial agent, characterized in that The bacterial agent contains the Geotrichum described in claim 1.

3. A fermentation broth, characterized in that: The fermentation liquid is obtained by fermenting the Geotrichum described in claim 1.

4. A composition, characterized in that The composition comprises the Geotrichum 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 Geotrichum described in claim 1; The fat substrate is rhodamine B.

9. A method for preparing an enzymatic hydrolysis product, characterized in that: The method comprises the steps of reacting an ester substrate with the Geotrichum described in claim 1; The ester substrate is tributyrin.

10. A method for preparing a degradation product, characterized in that: The method comprises the step of reacting PET with the Geotrichum described in claim 1.

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