Delftia delbrueckii and application thereof in degradation of PLA (polylactic acid) micro-plastics

Abstract

The invention relates to the technical field of environmental microorganisms, and discloses Delftia delbrueckii and application thereof in degradation of PLA (polylactic acid) microplastics. In particular discloses Delftia sp, the Delftia sp is Delftia sp DS1230, and the preservation number of the Delftia sp is CGMCC (China General Microbiological Culture Collection Center) No.30891 in the China General Microbiological Culture Collection Center. The strain or the microbial agent prepared from the strain can reduce the valence of polylactide, and can be used for industrial production.

Description

Technical Field

[0001] The present invention relates to the technical field of environmental microorganisms, and in particular to Delftia and application thereof in degrading PLA microplastics. Background Art

[0002] The official report released by the United Nations Environment Programme in 2023 clearly pointed out that the annual production of plastics in the world has exceeded the critical threshold of 430 million tons. Among them, about two-thirds of plastic products are of short-term use type and then quickly converted into waste. Given that traditional petroleum-based plastics are extremely difficult to degrade in the natural environment due to their CC skeleton structure, large molecular weight, strong covalent bond stability and high hydrophobicity, this has become an environmental problem that needs to be solved urgently.

[0003] In the context of pursuing sustainable development, bio-based plastics have received widespread attention as a potential alternative. In particular, PLA (polylactic acid), which is derived from renewable resources (such as wheat, corn, etc.) and is biodegradable, has become one of the most watched materials with broad development prospects.

[0004] In the field of green end-of-life management of plastics, microbial (enzyme) degradation technology occupies a vital position. PLA is mainly degraded by bio-enzymes through anaerobic digestion and composting in the soil. However, due to the high mechanical strength of PLA, its degradation rate is relatively slow compared to other biodegradable plastics, and the distribution of degrading bacteria is not yet widespread, so the natural degradation process may last for several years.

[0005] It is worth noting that when PLA is subjected to external forces (such as crushing, wear, weathering, erosion, biochemical reactions, etc.), it will produce microplastics with a diameter of less than 5mm. These microplastics have been listed as new pollutants by my country's Ministry of Ecology and Environment due to their environmental persistence and potential biological toxicity, and corresponding major scientific and technological projects have been launched for management and response.

[0006] Therefore, screening out bacterial strains that can effectively degrade PLA microplastics has become an urgent need. Summary of the invention

[0007] In view of this, the present invention proposes a Delftia sp. and its application in degrading PLA microplastics. The applicant has successfully isolated a genus named G-Delftia sp. (Delftiasp.DS1230) from the mulch film covering the farmland soil with PLA as the raw material. The genus can use PLA as a carbon source, grow and survive under the conditions of 25°C and 150rpm, and has a positive effect on the decomposition of PLA. After 28 days of cultivation, the weight loss rate of PLA reached 0.98%.

[0008] The present invention provides a Delftia sp., wherein the Delftia sp. is Delftia sp. DS1230, and its classification name is Delftia sp. It has been deposited in the General Microbiological Center of China National Committee for Microbiological Culture Collection on June 7, 2024, with a deposit address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and a deposit number of CGMCC No. 30891.

[0009] The invention discloses a bacterial agent, which contains the Delftia bacteria.

[0010] The present invention discloses an application of the Delftia bacteria or the bacterial agent, and the application is any one of the following:

[0011] A1) Application in degradation of polylactide;

[0012] A2) Application in the preparation of degradable polylactide products.

[0013] The present invention discloses an application of the Delftia bacteria or the bacterial agent, and the application is any one of the following:

[0014] B1) Application in assisting degradation of polylactide;

[0015] B2) Application in the preparation of assisted degradation polylactide products.

[0016] The present invention discloses an application of the Delftia bacteria or the bacterial agent, and the application is any one of the following:

[0017] C1) Application in improving the degradation rate of polylactide;

[0018] C2) Application in the preparation of products with improved degradation rate of polylactide.

[0019] The invention discloses a method for degrading polylactide, which comprises the step of using the Delftia bacteria and / or the bacterial agent to degrade the polylactide.

[0020] The invention discloses a method for preparing a polylactide degradation product. The method comprises the step of degrading the polylactide prepared by the method as a raw material.

[0021] The present invention discloses an application of the above method, and the application is any one of the following:

[0022] D1) Application in degradation of polylactide;

[0023] D2) Application in the preparation of degradable polylactide products.

[0024] The present invention discloses an application of the above method, and the application is any one of the following:

[0025] E1) Application in assisting degradation of polylactide;

[0026] E2) Application in the preparation of assisted degradation polylactide products.

[0027] The invention discloses a method for preparing a bacterial agent, which comprises the steps of using the Delftia bacteria as a component of the bacterial agent to obtain the bacterial agent.

[0028] The present invention conducts research on the level of microbial degradation of PLA, which has practical value for the treatment and application of PLA microplastics, helps to reduce its accumulation in soil and water bodies, and reduces the impact on the ecosystem; further promotes sustainable development, and also allows PLA to be more comprehensively and objectively understood and evaluated.

[0029] The present invention conducts research on the level of microbial degradation of PLA, which has practical value for the treatment and application of PLA microplastics, helps to reduce its accumulation in soil and water bodies, and reduces the impact on the ecosystem; further promotes sustainable development, and also allows PLA to be more comprehensively and objectively understood and evaluated.

[0030] Compared with the prior art, the invention has the following beneficial effects:

[0031] 1. We isolated a never-before-reported strain of PLA-degrading bacteria, Delftiasp. DS1230, and evaluated its potential to degrade PLA microplastics, aiming to alleviate the problem of PLA microplastic pollution and achieve sustainable development goals.

[0032] 2. The existing technology still pays much attention to the degradation of petroleum-based microplastics such as polyethylene, polypropylene, and polystyrene, but there is less research on PLA microplastics. The present invention explores the difficulty and complexity of its actual degradation from the perspective of biodegradable plastic PLA, which arouses social attention. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] 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:

[0034] Figure 1 This is a phylogenetic tree of Delftia sp. DS1230 drawn using MEGA software after 16s RNA sequencing and alignment;

[0035] Figure 2Schematic diagram of the growth curve of Delftia sp. DS1230 inoculated in M9 liquid medium containing 100 mg / L PLA;

[0036] Figure 3 This is the change in the mass of PLA before and after 28 days of degradation by Delftia sp. DS1230, from the initial 20.5 mg to 20.3 mg, with a degradation rate of 0.98%. DETAILED DESCRIPTION

[0037] 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.

[0038] Example 1 Identification of the Isolating Agent of Delftia

[0039] A piece of farmland soil from Yuncheng City, Shanxi Province was covered with mulch film, and the leaching liquid of the mulch film was inoculated into M9 minimal inorganic salt solid medium containing PLA powder. The main components of M9 medium are: 17.1g / LNa2HPQ4.12H2O, 3g / LKH2PO4, 0.5g / L NaCl, 1g / L NH4Cl, 1mM MgSO4, 1mMCaCl and trace elements, and the rest is water. 18g / L agar was added to the solid medium. It was grown in a constant temperature incubator at 25℃, and a strain of the genus was purified and isolated. After full-genome sequencing, it was identified as Delftia sp. The GenBank number of the full-genome sequence is CP157763. Among them, the 16sRNA sequence is shown in Sequence 1, and it is named Delftia sp.DS1230. The phylogenetic tree is constructed as shown in Figure 1 . Then, it was cultured in a shaker in liquid M9 medium containing PLA particles (Suzhou Shaoqian Plastic Chemical Company, molecular weight of about 50,000Da). The experiment proved that this strain can grow and proliferate with PLA as the only carbon source, increasing from the initial OD600 value of 0.008 to 0.2, participating in the degradation of PLA, with a degradation rate of 0.98%. The above target strain was deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration through patent procedures on June 7, 2024. The deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 30891.

[0040] Sequence 1 is as follows:

[0041]

[0042] Example 2 Application of Delftia

[0043] M9 solid plate containing 100 mg / L PLA: peptone 10 g / L (Beijing Aoboxing Biotechnology Co., Ltd., Catalog No.: 01-001), yeast extract 5 g / L (Beijing Solarbio Science & Technology Co., Ltd., Catalog No. 119-44-8), sodium chloride 10 g / L (Tianjin Guangfu Technology Development Co., Ltd., Catalog No.: 20120208), agar 18 g / L (Beijing Aoboxing Biotechnology Co., Ltd., Catalog No.: 01-023), and the rest is water.

[0044] M9 liquid medium containing 100 mg / L PLA: 17.1 g / L Na2HPQ4.12H2O (Shanghai MacLean Biochemical Technology Co., Ltd., Catalog No.: 10039-32-4), 3 g / L KH2PO4 (Sinopharm Chemical Reagent Co., Ltd., Catalog No.: 20150212), 0.5 g / L NaCl (Tianjin Guangfu Technology Development Co., Ltd., Catalog No.: 20120208), 1 g / L NH4Cl (Shanghai MacLean Biochemical Technology Co., Ltd., Catalog No.: 12125-02-9), 1 mM MgSO4 (Beijing Chemical Plant, Catalog No.: 20150702), 1 mM CaCl (Shanghai MacLean Biochemical Technology Co., Ltd., Catalog No.: 10043-52-4), and the rest is water.

[0045] Delftia sp. DS1230 was activated by streaking on an M9 solid plate containing 100 mg / L PLA powder, and cultured in a constant temperature incubator at 28°C for 18-24 hours. A single colony on the plate was inoculated into a conical flask containing 200 mL of M9 liquid culture medium containing 100 mg / L PLA. The culture temperature was 25°C, the shaker speed was 150 rpm, and the culture time was 28 days (cultivation ended). During this period, the growth of bacteria was monitored and represented by the optical density OD600 value of the bacterial suspension under a beam of light with a wavelength of 600 nanometers (the results are shown in FIG. 2 ). Figure 2 After the culture, the initial PLA mass and the PLA mass after degradation were measured (the results are shown in Figure 3 As shown, Figure 3 In the figure, initial is the initial PLA mass, and after degradation is the PLA mass after degradation), and the weight loss rate change of PLA is calculated. The weight loss rate change of PLA is 0.98%.

[0046] Initial PLA mass: The PLA mass obtained by weighing on an analytical balance.

[0047] Mass of PLA after degradation: After the cultivation is completed, the fermentation product is soaked in SDS detergent for 4 h, and intermittent ultrasonication is performed with an ultrasonic instrument to remove bacteria attached to the surface. Then, it is repeatedly rinsed with dd water three times, placed in a 50°C oven to dry overnight, and the mass is weighed using an analytical balance.

[0048] Changes in weight loss rate of PLA:

[0049] 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. Delftia, characterized in that The Delftia sp. is Delftia sp. DS1230, and its deposit number in the General Microbiological Center of China National Microbiological Culture Collection is CGMCC No. 30891.

2. A bacterial agent, characterized in that: The bacterial agent contains the Delftia sp. according to claim 1.

3. Use of the Delftia bacteria according to claim 1 or the bacterial agent according to claim 2, characterized in that: The application is any of the following: A1) Application in degradation of polylactide; A2) Application in the preparation of degradable polylactide products.

4. Use of the Delftia bacteria according to claim 1 or the bacterial agent according to claim 2, characterized in that: The application is any of the following: B1) Application in assisting the degradation of polylactide; B2) Application in the preparation of assisted degradation polylactide products.

5. Use of the Delftia bacteria according to claim 1 or the bacterial agent according to claim 2, characterized in that: The application is any of the following: C1) Application in improving the degradation rate of polylactide; C2) Application in the preparation of products with improved degradation rate of polylactide.

6. A method for degrading polylactide, characterized in that: The method comprises the step of using the Delftia bacteria described in claim 1 and / or the bacterial agent described in claim 2 to degrade polylactide.

7. A method for preparing a polylactide degradation product, characterized in that: The method comprises the step of degrading the polylactide prepared by the method of claim 6 as a raw material.

8. Use of the method according to claim 6 or 7, characterized in that: The application is any of the following: D1) Application in degradation of polylactide; D2) Application in the preparation of degradable polylactide products.

9. Use of the method according to claim 6 or 7, characterized in that: The application is any of the following: E1) Application in assisting degradation of polylactide; E2) Application in the preparation of assisted degradation polylactide products.

10. A method for preparing a bacterial agent, characterized in that: The method comprises the step of using the Delftia bacteria described in claim 1 as a component of the bacterial agent to obtain the bacterial agent.

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