Acinetobacter Guilin and application of acinetobacter Guilin in degradation of PBAT / PLA plastic

Abstract

The acinetobacter Guilin is preserved in the China Center for Type Culture Collection (CCTCC), the preservation date is October 17, 2024, and the preservation number of the strain is CCTCCNO: M 20242235. The acinetobacter Guilin has the advantages that the acinetobacter Guilin can be used for degrading PBAT / PLA plastics, and the acinetobacter Guilin can be used for degrading PBAT / PLA plastics; according to the invention, effective degradation of PBAT / PLA plastic can be realized.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and in particular to Acinetobacter guillouiae and application thereof in degrading PBAT / PLA plastics. Background Art

[0002] Polylactic acid (PLA), also known as polylactide, is named because it is mostly prepared by ring-opening polymerization of lactic acid cyclic dimer (i.e. lactide). It has good transparency, gloss, antibacterial properties, acid and alkali resistance and other characteristics. PBAT is a copolymer of butylene adipate and butylene terephthalate. It has good ductility, elongation at break, heat resistance and impact resistance, and excellent biodegradability. It has good film-forming properties and is usually blended and modified with PLA resin to form the end product - PBAT / PLA plastic, which has been widely used in the field of plastic packaging and agricultural mulch. There are many uncontrollable factors in the biodegradation process of PBAT / PLA plastics. Affected by multiple factors such as temperature, humidity, and bacterial species, PBAT / PLA plastics cannot be completely decomposed in many natural environments. Summary of the invention

[0003] The object of the present invention is to provide a strain of Acinetobacter guilinensis and application thereof in degrading PBAT / PLA plastics.

[0004] To achieve the above-mentioned purpose and other related purposes, the technical solution provided by the present invention is: a strain of Acinetobacter guilinensis, wherein the Acinetobacter guilinensis is Acinetobacter guilinensis P12-2, which is preserved in the China Center for Type Culture Collection (CCTCC), the preservation date is October 17, 2024, and the strain preservation number is CCTCCNO: M 20242235.

[0005] To achieve the above-mentioned purpose and other related purposes, the technical solution provided by the present invention is: the use of the Guilin Acinetobacter in the degradation of PBAT / PLA plastics.

[0006] The preferred technical solution is: comprising the following steps:

[0007] Step 1: Strain activation and preservation

[0008] The preserved Acinetobacter guilinensis strains were streaked on a 2216E solid culture medium, cultured at 35-40°C for 2-5 days, a single colony was picked up with an inoculation needle and inoculated into a 2216E liquid culture medium, and cultured on a shaker at 35-40°C and 100-200 rpm for 20-30 hours; then inoculated into the 2216E liquid culture medium at an inoculum size of 1-3%, and activated to obtain an activated bacterial solution;

[0009] Step 2: Degradation of PBAT / PLA plastics

[0010] Sterilize PBAT / PLA plastic and add it to the culture medium, then culture it at 35-40℃ to degrade PBAT / PLA plastic

[0011] The preferred technical solution is: 1L of 2216E liquid culture medium contains: 5.0g peptone, 1.0g yeast extract powder, 0.1g FeC6H5O7, 19.45g NaCl, 5.98g MgCl2, 3.24g Na2SO4, 1.8g CaCl2, 0.55g KCl, 0.16g Na2CO3, 0.08g KBr, 0.034g SrCl2, 0.022g H3BO3, 0.004g Na2SiO3, 0.0024g NaF, 0.0016g NH4NO3, and 0.008g Na2HPO4; the pH value is 7.6±0.2 (25°C).

[0012] b1L of 2216E solid culture medium contains: 5.0g of peptone, 1.0g of yeast extract powder, 0.1g of FeC6H5O7, 19.45g of NaCl, 5.98g of MgCl2, 3.24g of Na2SO4, 1.8g of CaCl2, 0.55g of KCl, 0.16g of Na2CO3, 0.08g of KBr, 0.034g of SrCl2, 0.022g of H3BO3, 0.004g of Na2SiO3, 0.0024g of NaF, 0.0016g of NH4NO3, 0.008g of Na2HPO4, and 16g of agar; the pH value is 7.6±0.2 (25℃).

[0013] The preferred technical solution is: the culture medium in step 2 is 2216E solid culture medium or 2216E liquid culture medium.

[0014] Due to the application of the above technical solution, the present invention has the following advantages compared with the prior art:

[0015] The present invention can achieve effective degradation of PBAT / PLA plastics. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is a bar graph showing the weight loss rate of PBAT / PLA plastics degraded by strain Acinetobacter guillouiae.

[0017] Figure 2 This is the growth curve of strain Acinetobacter guillouiae.

[0018] Figure 3The amylase activity of strain Acinetobacter guillouiae in plastics with and without PBAT / PLA added.

[0019] Figure 4 The protease activity of strain Acinetobacter guillouiae in plastics with and without PBAT / PLA added.

[0020] Figure 5 Scanning electron microscopy results of the degradation of PBAT / PLA plastics after strain Acinetobacter guillouia was added to the soil environment.

[0021] Figure 6 To examine the degradation of PBAT / PLA plastics by strain Acinetobacter guillouiae in natural environments. DETAILED DESCRIPTION

[0022] The following is a description of the implementation of the present invention by means of specific embodiments. Those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

[0023] See also Figure 1-6 . It should be noted that the structures, proportions, sizes, etc. illustrated in the drawings in this specification are only used to match the contents disclosed in the specification for people familiar with this technology to understand and read, and are not used to limit the limiting conditions for the implementation of the present invention, so they have no substantial technical significance. Any modification of the structure, change in the proportional relationship, or adjustment of the size should still fall within the scope of the technical content disclosed by the present invention without affecting the effects and purposes that can be achieved by the present invention. At the same time, the terms such as "upper", "lower", "left", "right", "middle" and "one" quoted in this specification are only for the convenience of description, and are not used to limit the scope of the implementation of the present invention. Changes or adjustments in their relative relationships should also be regarded as the scope of the implementation of the present invention without substantially changing the technical content.

[0024] Deposit of biological material:

[0025] Acinetobacter guillouiae P12-2, deposited in China Center for Type Culture Collection (CCTCC), deposited on October 17, 2024, with a strain deposit number of CCTCCNO: M 20242235. The deposit address is: China Center for Type Culture Collection, Wuhan University, Wuhan, China.

[0026] Unless otherwise specified, the reagents or materials described in the following examples are commercially available.

[0027] Example 1: A strain of Acinetobacter guilinensis and its application in the degradation of PBAT / PLA plastics

[0028] 1.1 Experimental strains

[0029] The source of the strain is screened from marine mud.

[0030] 1.2 Primary screening culture medium

[0031] LB solid medium: peptone 10.0 g, NaCl 10.0 g, yeast extract 5.0 g, agar 16.0 g, ultrapure water 1.0 L; LB liquid medium: peptone 10.0 g, NaCl 10.0 g, yeast extract 5.0 g, ultrapure water 1.0 L.

[0032] 1.3 Separation method

[0033] Take the marine mud sample into a sterilized 50ml centrifuge tube, add 20mL of sterilized deionized water, and place it in a 37℃ constant temperature shaking incubator at 200rpm for 12h. Shake thoroughly and shake well. Use a pipette to draw 200μL of sample, inoculate it into LB liquid culture medium, place it in a 37℃ incubator, and culture it for 1-2 days. Pipette 100μL of LB liquid culture medium culture solution onto LB solid culture medium for coating and culture for 2-3 days. The coated solid culture medium was sent to a sequencing company for strain identification, and a strain of Acinetobacter guillouiae was identified.

[0034] 1.4 Culture medium

[0035] 2216E liquid culture medium (1L): peptone 5.0g, yeast extract powder 1.0g, FeC6H5O7 0.1g, NaCl 19.45g, MgCl2 5.98g, Na2SO4 3.24g, CaCl2 1.8g, KCl 0.55g, Na2CO3 0.16g, KBr 0.08g, SrCl20.034g, H3BO3 0.022g, Na2SiO3 0.004g, NaF 0.0024g, NH4NO3 0.0016g, Na2HPO40.008g, pH 7.6±0.2 (25℃).

[0036] 2216E solid culture medium (1L): peptone 5.0g, yeast extract powder 1.0g, FeC6H5O7 0.1g, NaCl 19.45g, MgCl2 5.98g, Na2SO4 3.24g, CaCl2 1.8g, KCl 0.55g, Na2CO3 0.16g, KBr 0.08g, SrCl20.034g, H3BO3 0.022g, Na2SiO3 0.004g, NaF 0.0024g, NH4NO3 0.0016g, Na2HPO40.008g, 16g agar, pH 7.6±0.2 (25℃).

[0037] 1.2.1 Strain activation and preservation

[0038] The strains frozen in -80℃ glycerol storage were streaked on 2216E solid medium, and cultured in a 37℃ constant temperature incubator for 3 days. A single colony was picked up with an inoculation needle and inoculated into 3mL, 2216E liquid medium, and cultured on a shaker at 37℃ 160rpm for 24h. Then 2% of the inoculum was inoculated into 3mL, 2216E liquid medium, and activated for two generations. The activated strains were maintained at a ratio of glycerol: bacterial liquid = 1:2, and a portion of the bacterial liquid after two activations was taken to measure the OD 600 value.

[0039] 1.2.2 Growth curve determination

[0040] Take 12mL of liquid culture medium into a 50mL centrifuge tube, label it with the corresponding strain number, inoculate 100μL of activated bacterial liquid into the culture medium, place it in a 37℃ shaking incubator for culture, measure the OD value every 2h, and draw the growth curve of different strains.

[0041] like Figure 1 As shown in the figure, in the early stage of the culture process from 0 to 4 hours, the growth rate of the strain Acinetobacter guillouiae was relatively slow, and the strain was in the growth retardation period. In the middle stage of the culture process from 4 to 10 hours, the growth rate of the strain was significantly accelerated. After 12 hours, the growth of the strain gradually stabilized, and the OD value of the strain remained basically unchanged and reached the stable period of growth. After that, the concentration of the strain showed a downward trend.

[0042] 1.2.3 Determination of weight loss rate

[0043] After UV sterilization, the plastic was spread on a solid culture medium coated with bacterial solution. In order to monitor the weight change of the plastic during the degradation process, samples were taken on the 0th, 2nd, 4th, 6th and 8th days of the experiment to determine the weight loss rate of the bio-based plastic. The PBAT / PLA plastic in the culture medium was taken out with tweezers and repeatedly rinsed with double distilled water to remove the microorganisms attached to the surface of the PBAT / PLA plastic. Finally, the washed PBAT / PLA plastic was dried in an oven at 37°C overnight and weighed after cooling to room temperature. The weight loss rate of PBAT / PLA plastic is calculated by the following formula:

[0044]

[0045] Where, m0 is the initial mass; m1 is the mass after degradation.

[0046] like Figure 2 As shown in the figure, the weight loss rate of PBAT / PLA plastic was measured on the 8th day, and the weight loss rate of the strain Acinetobacterguillouiae reached 24.4%, while the weight loss rate of the control group was 15.8%. Compared with the control group, the weight loss rate of the strain was better than that of the control group, and the difference was more significant, indicating that it has a better promoting effect on the degradation of PBAT / PLA plastic.

[0047] 1.2.4 Determination of amylase activity

[0048] Take 10mL of 1% starch solution and add it to a test tube. Keep it in a 37℃ water bath for 10 minutes, then add 1mL of degradation fermentation liquid to each tube and keep it in a 37℃ constant temperature water bath for 10 minutes. Finally, add 1mL of 1M hydrochloric acid to terminate the reaction. Then take each test tube and add 10mL of iodine solution. Take 1mL of the reaction-terminated mixture and add it to the 10mL iodine solution test tube and shake well. Take out the completely reacted liquid, measure the absorbance under 660nm wavelength light, and record the data. The control group does not add enzyme solution, but adds the corresponding volume of water. Calculation of amylase activity in each mL of fermentation liquid:

[0049]

[0050] Wherein, ΔA is the absorbance difference between the sample and the control group; t is the enzyme action time (10 min in this experiment); w is the amount of enzyme used in the reaction.

[0051] like Figure 3 As shown, the activity of strain Acinetobacter guillouiae was significantly higher than that of the control group, and the amylase activity of the plastic with PBAT / PLA added was significantly higher than that of the plastic without PBAT / PLA added.

[0052] 1.2.5 Protease activity assay

[0053] Studies have shown that protease activity can reflect the response of strains to PBAT / PLA plastics, and the degradation of strains to PBAT / PLA plastics can be indirectly detected by detecting protease activity. Keep 5% TCA solution and 1% casein solution at 37°C. Take four 15mL stoppered test tubes, inhale 0.20ml of fermentation liquid into each of two test tubes, marked as group A; inhale 0.40ml of fermentation liquid into each of the other two test tubes, marked as group B. Then use 0.2mol / L phosphate buffer to make the volume 2.00mL. Inhale 6.00mL of 5% TCA solution into each test tube in groups A and B, and keep the above four test tubes in a 37°C water bath. Inhale 2.00mL of 1% casein solution in each test tube, keep it at 37°C for 10min (accurate timing), and then inhale 6.00mL of 5% TCA solution into the other test tube of groups A and B. Take the test tube out of the water bath and place it at room temperature for 1h. At a wavelength of 280nm, the absorbance of the fermentation broth just after adding 5% TCA solution is 0. The absorbance of the liquid after the fermentation broth reacts for ten minutes is measured. Calculation of protease activity in each mL of fermentation broth:

[0054]

[0055] Wherein, ΔA is the absorbance difference between the sample and the blank; t is the enzyme action time (10 min in this experiment); w is the amount of enzyme used in the reaction.

[0056] like Figure 4 As shown, the activity of strain Acinetobacter guillouiae was significantly higher than that of the control group, and the protease activity of the plastic with PBAT / PLA added was significantly higher than the amylase activity of the plastic without PBAT / PLA added.

[0057] 1.2.6 Scanning electron microscopy observation

[0058] Relying on a desktop electron scanning microscope (COXEM EM-30Plus), the degraded PBAT / PLA plastic was cut to a suitable size, the degraded fragments were clamped, and the soil on the surface of the PBAT / PLA plastic was rinsed with deionized water to remove the PBAT / PLA plastic. The PBAT / PLA plastic fragments were spread on aluminum foil and then placed in a fume hood to dry naturally. Then the PBAT / PLA plastic samples were sprayed with gold, and the sample height was adjusted during placement. When scanning and taking pictures, the field of view was found at low magnification, the focus was adjusted at high magnification, and then the pictures were taken in order from high magnification to low magnification.

[0059] like Figure 5As shown, combined with the above results, the strain Acinetobacter guillouiae can efficiently degrade PBAT / PLA plastics, and it was added to the soil environment for observation. Comparison of electron microscopy images showed that after one week, there were many obvious cracks on the surface of the PBAT / PLA plastics in the bacterial group, while only fine cracks appeared in the natural group, indicating that the application of the screened strains can accelerate the degradation of PBAT / PLA plastics in the natural environment. After 28 days of degradation, there were no flat parts on the surface of the PBAT / PLA plastics in the natural group, but no large-scale fracture pits appeared, while the bacterial group was obviously degraded, and many fracture pits appeared on the surface of the PBAT / PLA plastics. 1.2.7

[0061] like Figure 6 As shown in the figure, the degradation of PBAT / PLA plastics by the screened high-efficiency degradation strain Acinetobacter guillouia was tested in the natural environment. The weight loss rate of the experimental group with the strain was significantly higher than that of the natural group, and the weight loss rate of the group with the strain exceeded 50% after 28 days, indicating that it does promote the degradation of PBAT / PLA plastics.

[0062] The above description is only used to explain the preferred embodiments of the present invention, and is not intended to limit the present invention in any form. Therefore, any modifications or changes made to the present invention under the same inventive spirit should still be included in the scope of protection intended by the present invention.

[0063] References

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Claims

1. A strain of Acinetobacter guilinensis, characterized in that: The Guilin Acinetobacter is Guilin Acinetobacter P12-2, which is preserved in the China Center for Type Culture Collection (CCTCC), the preservation date is October 17, 2024, and the strain preservation number is CCTCCNO: M 20242235.

2. Application of Acinetobacter guilinensis according to claim 1 in degrading PBAT / PLA plastics.

3. The use of Acinetobacter guilinensis in the degradation of PBAT / PLA plastics according to claim 2, characterized in that: The following steps are involved: Step 1: Strain activation and preservation The preserved Acinetobacter guilinensis strains were streaked on a 2216E solid culture medium, cultured at 35-40°C for 2-5 days, a single colony was picked up with an inoculation needle and inoculated into a 2216E liquid culture medium, and cultured on a shaker at 35-40°C and 100-200 rpm for 20-30 hours; then inoculated into the 2216E liquid culture medium at an inoculum size of 1-3%, and activated to obtain an activated bacterial solution; Step 2: Degradation of PBAT / PLA plastics The PBAT / PLA plastic is sterilized and added to the culture medium, and then cultured at 35-40°C to degrade the PBAT / PLA plastic.

4. The use of Acinetobacter guilinensis in the degradation of PBAT / PLA plastics according to claim 3, characterized in that: 1L of 2216E liquid culture medium contains: 5.0g of peptone, 1.0g of yeast extract powder, 0.1g of FeC6H5O7, 19.45g of NaCl, 5.98g of MgCl2, 3.24g of Na2SO4, 1.8g of CaCl2, 0.55g of KCl, 0.16g of Na2CO3, 0.08g of KBr, 0.034g of SrCl2, 0.022g of H3BO3, 0.004g of Na2SiO3, 0.0024g of NaF, 0.0016g of NH4NO3, and 0.008g of Na2HPO4; pH value is 7.6±0.2 (25℃).

5. The use of Acinetobacter guilinensis in the degradation of PBAT / PLA plastics according to claim 3, characterized in that: 1L of 2216E solid culture medium contains: 5.0g peptone, 1.0g yeast extract powder, 0.1g FeC6H5O7, 19.45g NaCl, 5.98g MgCl2, 3.24g Na2SO4, 1.8g CaCl2, 0.55g KCl, 0.16g Na2CO3, 0.08g KBr, 0.034g SrCl2, 0.022g H3BO3, 0.004g Na2SiO3, 0.0024g NaF, 0.0016g NH4NO3, 0.008g Na2HPO4, 16g agar; pH value is 7.6±0.2 (25℃).

6. The use of Acinetobacter guilinensis in the degradation of PBAT / PLA plastics according to claim 3, characterized in that: The culture medium in step 2 is 2216E solid culture medium or 2216E liquid culture medium.

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