Strain degrading polylactic acid plastic and method for enhancing degradation

Abstract

The application belongs to the technical field of microorganisms, and provides a polylactic acid (PLA) degrading bacterium and a method for enhancing degradation thereof. The bacterium belongs to Methylorubrum sp., and has been preserved in the China General Microbiological Culture Collection Center on March 9, 2023, with a preservation number CGMCC of 26776. The PLA degrading bacterium JX-1 obtained through separation and screening in the application enables PLA plastic mulch to achieve a high weight loss rate, realizes degradation of PLA plastic, and simultaneously obtains a method for accelerating degradation of PLA. The application has the advantage that the bacterium has the ability to degrade PLA and can enhance utilization of PLA under the stimulation of a carbon source. The bacterium is suitable for pollution remediation of PLA and PLA-based biodegradable materials in the environment, and provides a new strain resource for degradation of PLA biodegradable plastic and an optional method for enhancing remediation.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, and in particular relates to a PLA-degrading bacterium and a method for enhancing its application effect. Background Technology

[0002] In recent years, with the modernization of human society and the rapid development of industries such as fast food and express delivery, the demand and use of plastic products, especially single-use plastic products, have increased dramatically. Currently, most single-use plastic products (such as plastic bags and boxes) are discarded after a single use, leading to a significant increase in these non-degradable plastic wastes in the environment. Currently, the amount of recycled plastic in my country is very small, less than 5% of the total usage. Plastic incineration typically produces large amounts of toxic gases, which can severely damage the ozone layer. Landfill disposal of plastics also has insurmountable drawbacks because the physicochemical structure of most petroleum-based plastics is very stable, taking over 200 years to gradually decompose in soil, resulting in very slow degradation. Based on this, environmentally friendly biodegradable plastics that can replace petroleum-synthesized polymers have emerged. Currently, under the trend of sustainable development and circular economy development, biodegradable plastics are attracting increasing attention and importance, and have already demonstrated their value in many fields. Polylactic acid (PLA) is one of the most promising biodegradable plastics currently available.

[0003] PLA is a high-molecular-weight polymer synthesized from renewable resources, possessing relatively high thermal stability and crystallizable thermoplasticity. Its raw material is the monomer lactic acid, primarily derived from the starches of corn, cassava, and potatoes. The widespread use of PLA helps reduce greenhouse gas emissions and fossil fuel consumption; therefore, PLA is currently one of the most promising biodegradable plastics to replace petroleum-based materials and address "white pollution" and energy issues. Since its commercialization in the late 1990s, global bioplastic production has grown at an annual rate of 40%, with PLA accounting for over 20% of the market share. Compared to other aliphatic polyesters, PLA is easy to manufacture, non-toxic, harmless, and possesses good mechanical and physical properties, biodegradability, biocompatibility, and transparency, making it widely applicable in the automotive industry, biomedical equipment, clothing industry, and agricultural mulch films. However, with the expansion of PLA product commercialization, waste management has attracted widespread attention. Although PLA is a recognized biodegradable plastic, compared to other biodegradable plastics, fewer microorganisms exist in the environment to degrade PLA, meaning it takes several years to completely degrade in the natural environment. If PLA is not effectively treated, it still poses the potential to cause "white pollution," negatively impacting the environment and ecosystem. Therefore, ensuring PLA maintains stable properties throughout its lifespan while rapidly degrading after disposal has become a key focus for environmental researchers.

[0004] The degradation mechanisms of PLA mainly include thermal degradation, photodegradation, hydrolysis, and biochemical degradation. Among these, biochemical degradation does not produce other toxic or harmful substances and is currently the most effective and environmentally friendly treatment method. Screening for more effective PLA-degrading bacteria can provide more options for the remediation of PLA residual pollution. Currently, some microorganisms capable of degrading PLA have been isolated from soil, wastewater, and sludge both domestically and internationally. However, research on PLA-degrading bacteria in my country is limited. Domestically reported PLA-degrading bacteria include Pseudomonas sp., Lentzeawaywayandensis, and the fungus Cladosporium tenuissimum, but they generally suffer from low degradation rates, and PLA-degrading bacteria resources remain scarce. Obtaining more PLA-degrading bacteria and researching methods to improve their degradation rates are essential for the remediation of PLA-polluted environments. Summary of the Invention

[0005] The purpose of this invention is to provide a screening method for a PLA particle-degrading bacterium that has the ability to degrade PLA particles.

[0006] This invention is achieved through the following technical solution:

[0007] This invention first provides a polylactic acid degrading bacterium JX-1, which is classified as Methylorubrum sp. and has the accession number CGMCC NO.26776. The polylactic acid degrading bacterium JX-1 was isolated from farmland soil in Yingtan, Jiangxi Province.

[0008] The present invention further provides the application of the polylactic acid degrading bacterium JX-1 in the degradation of PLA.

[0009] Specifically, the polylactic acid degrading bacteria are used to degrade PLA in the environment.

[0010] Preferably, sucrose or soluble starch, a carbon source, is added during the degradation process to promote the degradation of PLA particles.

[0011] More specifically, a liquid bacterial agent was prepared by culturing polylactic acid degrading bacteria in LB medium and sprayed into the environment to degrade plastic residues.

[0012] The present invention also provides a bio-enhancing agent for degrading PLA, wherein the polylactic acid degrading bacteria described above are the active ingredients.

[0013] Specifically, it is obtained by culturing polylactic acid degrading bacteria in LB medium in the form of liquid inoculum, or by drying to obtain inoculum powder form.

[0014] Preferably, it also contains a carbon source, sucrose or soluble starch.

[0015] Compared with the prior art, the present invention has the following beneficial technical effects:

[0016] This invention isolated a PLA particle-degrading bacterium, JX-1, from farmland soil in Yingtan, Jiangxi Province. This strain exhibits excellent degradation efficiency for PLA. The degrading bacterium provided by this invention broadens our understanding of PLA particle-degrading bacteria. This invention also provides an application study of this strain in environmental PLA degradation and investigates the role of carbon sources in enhancing the degradation effect, providing a technical reference for the bioremediation of PLA in the environment. Attached Figure Description

[0017] Figure 1 This is a colony morphology diagram of the polylactic acid degrading bacterium JX-1 of the present invention;

[0018] Figure 2 The sequence alignment results of the polylactic acid degrading bacterium JX-1 of this invention are shown below.

[0019] Figure 3 The degradation rate of PLA particles by the polylactic acid degrading bacteria JX-1 of this invention at 15d, 30d, and 45d is shown.

[0020] Figure 4This is a scanning electron microscope observation of the surface morphology of PLA particles after 45 days of degradation by the polylactic acid degrading bacteria JX-1 of this invention.

[0021] Information on the preservation of biological materials:

[0022] The polylactic acid degrading bacterium JX-1 of the present invention was deposited: deposit date: March 9, 2023, depositary institution: China General Microbiological Culture Collection Center (CGMCC), address of depositary institution: Institute of Microbiology, Chinese Academy of Sciences, Beichen West Road, Chaoyang District, Beijing (postcode: 100101), its classification name: Methylorubrum sp., deposit number CGMCCNO.26776. Detailed Implementation

[0023] The present invention will be further described in detail below with reference to specific embodiments. These descriptions are for explanation purposes only and are not intended to limit the scope of the invention.

[0024] Example 1: Isolation and Screening of PLA Particle Degrading Bacteria

[0025] This invention isolates a PLA particle-degrading bacterium from farmland soil in Yingtan, Jiangxi Province, and classifies and names it JX-1.

[0026] This invention preserves PLA-degrading bacteria JX-1: deposit date: March 9, 2023, depositary institution: China General Microbiological Culture Collection Center (CGMCC), address of depositary institution: Institute of Microbiology, Chinese Academy of Sciences, Beichen West Road, Chaoyang District, Beijing, 100101, China, accession number: CGMCC26776.

[0027] The culture medium involved in this invention has the following formulation:

[0028] Inorganic salt culture medium with PLA as the sole carbon source ( / L): PLA particles (molecular weight approximately 50,000), approximately 1.6g, K2HPO4 5.8g, KH2PO4 4.5g, (NH4)2SO4 2.0g, MgCl2 0.16g, CaCl2 0.02g, Na2MoO4·2H2O 0.0024g, FeCl3 0.0018g, MnCl2·2H2O 0.0015g, pH=7.0; distilled water, 1000mL; pH 7.2–7.4.

[0029] LB medium: tryptone, 10g; yeast extract, 5g; NaCl, 10g; distilled water, 1000mL; pH 7.2–7.4; agar, 20g.

[0030] Farmland soil was collected from Yingtan, Jiangxi Province. 1 g of soil was added to 100 mL of liquid culture medium containing PLA as the sole carbon source (an inorganic salt culture medium). The soil was acclimated for one month in a constant-temperature shaker at 30°C using PLA particles as the sole carbon source, and then passaged for at least five generations. 1 mL of the enrichment solution was used to prepare 10... -1 Up to 10 -6 A series of bacterial suspensions at varying concentrations were streaked on LB medium and incubated at 30°C for 3-7 days. Single colonies were then picked and repeatedly inoculated onto LB medium for isolation and purification until a single bacterial strain was obtained. Colony morphology was then observed. Figure 1 ).

[0031] Example 2: Identification of the strain

[0032] 1. Morphological observation of the strain

[0033] Strain JX-1 was inoculated onto LB solid medium and cultured for 48 hours. After incubation, it formed round or nearly round, pale yellow colonies with regular edges, as shown in the attached image. Figure 1 As shown.

[0034] 2. Identification of the 16S rRNA gene sequence of the strain

[0035] The pure bacterial strain was inoculated onto LB liquid medium and cultured on a shaker at 30°C and 150 rpm for 1-2 days. DNA was extracted from the strain using the Tiangen bacterial genomic DNA extraction kit, employing universal primers 1429R (5'-CTACGGCTACCTTGTTACGA-3') and 27F (5'-AGAGTTTGATCCTGGCTCAG-3') for bacterial 16S rDNA. The PCR reaction mixture consisted of 50 μL: approximately 100 ng of DNA template, 1.0 μL each of 10 μmol / L 27F and 1429R, 4 μL of 10 mmol / L dNTPs, 5 μL of 10×PCR Buffer (2.5 mmol / L MgCl2), 0.6 μL of 2.5 U / μL Taq DNA polymerase, and sterile ultrapure water to a final volume of 50 μL. The reaction conditions were: 94℃ pre-denaturation for 10 min; 94℃ denaturation for 60 s, 58℃ annealing for 60 s, 72℃ extension for 90 s, 35 cycles; 72℃ extension for 10 min, and storage at 4℃. After detection by 10 g / L agarose gel electrophoresis, the PCR products were cloned and sequenced by Beijing Ruiboxingke Biotechnology Co., Ltd., yielding the 16S rRNA gene sequence of 1,366 bp as follows (SEQ ID NO: 1):

[0036] The results of the comparison with the microbial species identification database EZBioCloud (https: / / www.ezbiocloud.net / ) are attached. Figure 2 As shown, this strain shares the highest homology (99%) with the Methylorubrum populiBJ001 type strain (accession number: CP001029). Therefore, the degrading bacteria obtained by screening in this invention are polylactic acid degrading bacteria (Methylorubrum).

[0037] Therefore, the inventors deposited this strain at the Budapest Treaty International Collection Unit for Microorganisms: China General Microbiological Culture Collection Center (CGMCC). The deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. The deposit date is March 9, 2023, and the accession number is CGMCC NO.26776.

[0038] Example 2: Degradation of PLA particles using JX-1

[0039] Aseptic treatment of PLA particles: Soak in 100% alcohol for 4 hours in a 50ml sterile centrifuge tube, then rinse with sterile ultrapure water more than three times, place in a disposable clean culture dish, and freeze dry to constant weight. Weigh the particles.

[0040] In a 150 mL Erlenmeyer flask, strain JX-1, which uses PLA particles as the sole carbon source, was inoculated into 50 mL of basic inorganic salt medium. A blank control was set up with no bacterial culture added. Triple replicates were performed. The flasks were incubated at 150 rpm and 30 °C using a shaker. At 15, 30, and 45 days, PLA particles were removed using sterile forceps and collected. The particles were then soaked in 100% ethanol for 30 min, washed at least three times with ultrapure water, and dried at 65 °C for 12 h. The particles were accurately weighed, and the weight loss rate was determined to calculate the degradation ability of strain JX-1. The surface morphology of the PLA particles was also observed. The composition of the basic inorganic salt medium was the same as described in Example 1.

[0041] PLA particle weight loss rate (%) = (Initial PLA mass before treatment - PLA mass after inoculation treatment) / PLA mass before treatment × 100%

[0042] like Figure 3As shown, the degradation rate of PLA particles by the degrading bacterium JX-1 was 3.59% after 45 days of cultivation with only PLA particles (normal fermentation). The degradation rate was 4.46% with the addition of 0.5% common carbon source soluble starch, and 6.97% with the addition of 0.5% common carbon source sucrose powder. This demonstrates that both common carbon sources, sucrose and soluble starch, can significantly promote the degradation of PLA particles.

[0043] Measurement of Surface Morphology Changes in Polyethylene Film: After 45 days of bacterial inoculation, PLA particles were fixed and sputter-coated with gold. The microscopic changes in the surface morphology of the polyethylene film were observed using field emission scanning electron microscopy. The results are as follows: Figure 4 As shown, the surface of PLA particles becomes rough, with obvious erosion holes and clear cracks, proving that PLA has degraded.

[0044] This invention further enriches the microbial resources for degradable PLA plastics, providing fermentation materials for future degradable plastic pollution control.

Claims

1. A polylactic acid degrading bacterium, characterized in that, Its classification name is Methylorubrum sp., and it was deposited at the China General Microbiological Culture Collection Center on March 9, 2023, with the accession number CGMCC NO.26776.

2. The application of polylactic acid degrading bacteria as described in claim 1 as a biofortifier in the degradation of polylactic acid.

3. The application according to claim 2, characterized in that, The polylactic acid degrading bacteria described herein are applied to the degradation of polylactic acid in the environment.

4. The application according to claim 3, characterized in that, Carbon sources such as sucrose or soluble starch are added during the degradation process to promote the degradation of polylactic acid particles.

5. The application according to claim 3, characterized in that, Liquid bacterial agent was prepared by culturing polylactic acid degrading bacteria in LB medium and sprayed into the environment to degrade plastic residues.

6. A bio-fortifier for degrading polylactic acid, characterized in that, It uses polylactic acid degrading bacteria as described in claim 1 as its active ingredient.

7. The bio-fortifying agent for degrading polylactic acid as described in claim 6, characterized in that, It is obtained by culturing polylactic acid degrading bacteria in LB medium in the form of liquid inoculum, or by drying to obtain inoculum powder form.

8. The bio-fortifying agent for degrading polylactic acid as described in claim 6, characterized in that, It also contains added carbon sources such as sucrose or soluble starch.

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