Application of arthrobacter in plastic degradation

Abstract

The invention discloses application of arthrobacter in degradation of plastics, and relates to the technical field of microorganisms, the strain is named as arthrobacter sp.CY1, and is preserved in the China General Microbiological Culture Collection Center (CGMCC), the preservation number is CGMCC No.32460, and the preservation date is October 31, 2024. The arthrobacter sp. CY1 provided by the invention can breed and grow by taking the polyethylene plastic as a unique carbon, nitrogen and energy raw material, and the polyethylene plastic is degraded through metabolism; harmless biodegradation of the PE plastic can be achieved, and the method has very important significance in treatment of plastic waste.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to an application of Arthrobacter in degrading plastics. Background Art

[0002] While plastic products bring convenience to human life, they also pose severe challenges to the natural environment. Plastics come from a variety of sources, and different plastics have different properties and functions, including:

[0003] Polyethylene (PE) is a polymer compound formed by the polymerization of ethylene, and its molecular formula is (C2H4)n. PE and its derivatives are widely used in agricultural films, industrial packaging films, pharmaceutical and food packaging films, mechanical parts, daily necessities, building materials, wires, cable insulation, coatings and synthetic paper. Due to its widespread use, the accumulation of PE plastic waste in the environment has become a global environmental problem. Due to the characteristics of PE's corrosion resistance, impact resistance, wear resistance, low temperature resistance, acid and alkali resistance, and water resistance, the degradation and utilization of PE waste is very difficult.

[0004] At present, the treatment of PE plastic waste mainly relies on landfill and incineration. Physical landfill is a traditional treatment method, but because plastics are difficult to degrade, it will lead to long-term occupation of land resources and may cause groundwater pollution; incineration will produce harmful gases and dust, which will cause secondary pollution to the environment. PE plastic pollution remediation can be divided into physical methods, chemical methods and biological methods according to different treatment methods. Physical and chemical methods have the advantages of quick results, but the cost of remediation is expensive, the ecological effect is poor, and there is a risk of secondary pollution. Biological remediation can convert plastic components into harmless inorganic compounds (water and carbon dioxide). It has the advantages of low cost, high efficiency and eco-friendliness. It is regarded as a high-tech with broad prospects. It has been well developed at home and abroad in the past decade.

[0005] However, because PE has a wear-resistant, corrosion-resistant and water-resistant macromolecular structure, it is considered very difficult for microorganisms to biodegrade it. At present, the PE degradation strains reported in domestic and foreign studies on PE biodegradation are mainly Pseudomonas, Bacillus, Marinobacter, Selenobacter, Alkanophage, etc., but the sources of these degradation bacteria are mostly oceans, sewage and landfills, etc., and the ecological environment they are in is relatively complex, which makes the use of these degradation bacteria safe and there may be risks. Summary of the invention

[0006] In view of the above prior art, the purpose of the present invention is to provide an application of an Arthrobacter strain in degrading plastics. The Arthrobacter strain of the present invention has the ability to degrade PE; and the Arthrobacter strain of the present invention is isolated from the Ningxiang Demonstration Base, and is highly safe to use. The Arthrobacter strain of the present invention can achieve harmless biodegradation of plastics.

[0007] To achieve the above object, the present invention adopts the following technical solution:

[0008] The first aspect of the present invention provides a strain of Arthrobacter sp.CY1, which was deposited on October 31, 2024 in the General Microbiology Center of China Microorganism Culture Collection Committee (referred to as CGMCC, address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing), and its deposit number is CGMCCNO: 32460.

[0009] The Arthrobacter sp.CY1 was isolated from the soil that was long-term attached to plastic in the Ningxiang Demonstration Base in Changsha City, Hunan Province, my country (geographic coordinates 28.1603, 112.5557), and has the following characteristics:

[0010] The colonies are milky white, medium-sized, opaque and round; Gram staining is positive.

[0011] The second aspect of the present invention provides a bacterial agent containing the above-mentioned Arthrobacter sp.CY1.

[0012] Furthermore, in the above-mentioned bacterial agent, the Arthrobacter sp.CY1 can exist in the form of cultured live bacteria or fermentation liquid of live bacteria.

[0013] Furthermore, the fermentation broth is prepared by the following method:

[0014] The seed liquid of Arthrobacter sp.CY1 was inoculated into LB liquid medium and cultured at 28-37°C and 150-200r / min until the bacterial concentration OD 600 =0.8-1.

[0015] Furthermore, the above-mentioned bacterial agent may also contain other active ingredients, and those skilled in the art may determine the composition of other active ingredients according to the use effect of the bacterial agent.

[0016] Furthermore, the above-mentioned bacterial agent may also include a carrier, which includes but is not limited to peat, bamboo charcoal powder, kaolin, diatomaceous earth, bentonite, and sodium alginate.

[0017] Furthermore, the bacterial agent may be in the form of a liquid, powder, granule, etc.

[0018] The third aspect of the present invention provides the use of any of the above-mentioned Arthrobacter sp.CY1 or any of the bacterial agents in degrading plastics.

[0019] Furthermore, the plastic is polyethylene plastic (PE).

[0020] The fourth aspect of the present invention provides a method for degrading PE solution, comprising the following steps: mixing and contacting the PE solution to be degraded with any of the above-mentioned Arthrobacter sp.CY1 or any of the above-mentioned bacterial agents.

[0021] Furthermore, the composition of the PE solution is:

[0022] PE film 1g / L, 0.70g / LKH2PO4, 0.70g / LK2HPO4, 0.7g / LMgSO4·7H2O, 1.00g / L(NH4)2SO4 , 0.005g / LNaCl, 0.002g / LFeSO4·7H2O, 0.002g / LZnSO4·7H2O and 0.001g / LMnSO4·H2O.

[0023] The present invention also provides a method for degrading PE film, comprising the following steps: inoculating any of the above-mentioned Arthrobacter sp.CY1 into an inorganic salt liquid culture medium added with PE film, and culturing at 28°C-37°C.

[0024] The beneficial effects of the present invention include at least:

[0025] The present invention isolates a strain of Arthrobacter sp.CY1 capable of degrading PE from soil for the first time. The Arthrobacter sp.CY1 of the present invention can reproduce and grow with polyethylene plastic as the only carbon, nitrogen and energy raw material, degrade polyethylene plastic through metabolism, and can achieve harmless biodegradation of PE plastic, which is of great significance for the treatment of plastic waste. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is a colony picture of Arthrobacter sp.CY1.

[0027] Figure 2 This is the Gram staining image of Arthrobacter sp.CY1.

[0028] Figure 3 This is the phylogenetic tree of Arthrobacter sp.CY1.

[0029] Figure 4 This is the growth curve of Arthrobacter sp.CY1 in LB medium.

[0030] Figure 5This is the degradation of PE by Arthrobacter sp.CY1 in inorganic salt culture medium. DETAILED DESCRIPTION

[0031] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0032] In addition, the technical solutions between the various embodiments of the present invention can be combined with each other, but it must be based on the fact that ordinary technicians in the field can implement it. When the combination of technical solutions is contradictory or cannot be implemented, it should be deemed that such combination of technical solutions does not exist and is not within the scope of protection required by the present invention.

[0033] As mentioned above, due to the characteristics of PE molecular structure, its biodegradation is relatively difficult. There are relatively few strains reported that can be used for PE degradation, and most of them come from the ocean, sewage and landfills. The ecological environment in which they are located is relatively complex, which makes the use of these degrading bacteria safe and risky.

[0034] Based on this, in order to achieve harmless biodegradation of PE, the present invention continuously attempts to isolate new degradation strains. Finally, a strain of Arthrobacter sp.CY1 was isolated from cultivated soil with long-term plastic film attached in the Ningxiang demonstration base.

[0035] In addition, the present invention provides a method for preparing a PE solution for detecting degradation effects, wherein the composition of the PE solution is:

[0036] PE film 1g / L, 0.70g / L KH2PO4, 0.70g / LK2HPO4, 0.7g / LMgSO4·7H2O, 1.00g / L (NH4)2SO4, 0.005g / L NaCl, 0.002g / L FeSO4·7H2O, 0.002g / L ZnSO4·7H2O and 0.001g / LMnSO4·H2O.

[0037] The present invention found that the Arthrobacter sp.CY1 strain can significantly degrade PE film under liquid culture conditions for a relatively short period of time (30 days), causing the weight loss of the PE film to be nearly 2.4%; and the weight loss of the PE film can be about 3.3% in 90 days. The degradation effect and speed are both very significant, which is a major breakthrough in the exploration of the function of Arthrobacter.

[0038] Therefore, the Arthrobacter sp.CY1 of the present invention has great application value in plastic degradation, and thus the present invention is proposed.

[0039] In order to enable those skilled in the art to more clearly understand the technical solution of the present application, the technical solution of the present application will be described in detail below in conjunction with specific examples. If the specific experimental conditions are not specified in the examples, they are usually in accordance with conventional conditions or the conditions recommended by the reagent company; the reagents, consumables, etc. used in the following examples, unless otherwise specified, can be obtained through commercial channels.

[0040] The solution proposed by the present invention is specifically described below through specific embodiments:

[0041] Example 1: Isolation and identification of strains

[0042] 1. Isolation of strains:

[0043] Soil attached to plastic was collected from the Ningxiang Demonstration Base in Changsha, Hunan Province, which was covered with PE mulch for a long time. After removing the surface soil, the 0-30cm soil layer containing plastic was collected with sterile utensils. Weigh 1.5g of fresh soil and transfer it to a 250mL conical flask containing 100mL of 0.9% sterile saline (9g NaCl diluted to 1000mL). Shake it at 180rpm, 28℃, and oscillate for 2h. Take the upper clear liquid as the bacterial suspension. Dilute the prepared bacterial suspension according to a certain proportion, take 1mL of the bacterial suspension and transfer it to a 250mL conical flask containing 100mL of LCFBM (liquid carbon-free culture medium) and a sterile PE film. The conical flask was shaken and cultured at 28℃ and 180rpm. After 15 days of culture, the PE film was transferred to a new culture medium for the next enrichment stage. The enrichment test included 6 inoculation stages, a total of 90 days of culture, and 3 repeated treatments. Control treatment: PE without microbial culture (no inoculation of bacterial suspension). On the 90th day, the membrane was taken out and placed in sterile phosphate buffered saline (PBS) with a pH of 7.4 and a concentration of 0.01 M, and then shaken and washed to prepare a bacterial suspension.

[0044] Take 5 μL of the screened bacterial suspension and spread it on NB solid medium (beef extract 3.0 g / L, peptone 5.0 g / L, sodium chloride 5.0 g / L, agar 15.0 g / L) for streaking, culture in a biochemical incubator at 28°C for 3-5 days, separate and purify 3-5 times until a single colony with consistent morphology grows. Store the purified strain obtained by screening at -20°C.

[0045] 2. Identification of strains

[0046] (1) Gram staining and morphological identification:

[0047] The purified Arthrobacter sp.CY1 was subjected to Gram staining, and the result was positive ( Figure 2 ), the colonies of this fungus are milky white, medium-sized, opaque and round ( Figure 1 ).

[0048] (2) Molecular identification:

[0049] In order to determine the phylogenetic status of Arthrobacter sp.CY1, the isolated strain was analyzed by 16S rDNA conservative sequence analysis. The obtained sequence results were compared with the BLAST search results of the National Center for Bioinformation (NCBI) in the United States to determine that the isolated strain was Arthrobacter sp. and was named CY1. The phylogenetic tree is shown in Figure 3 .

[0050] The above strains can grow well in LB medium (tryptone 10g / L, yeast extract 5g / L, NaCl 10g / L), and their growth curves are shown in Figure 4 Arthrobacter sp.CY1 bacteria originate from the natural environment. When released into the natural environment, they are harmless to humans, animals and plants and do not pollute the environment.

[0051] The laboratory preservation method of the obtained Arthrobacter sp.CY1 is as follows:

[0052] The short-term preservation method is to inoculate the strain onto an LB solid plate. The LB solid culture medium formula is: 10 g / L tryptone, 5 g / L yeast extract, 5 g / L NaCl, 15 g / L agar, pH to 7.2. After the strain grows well, it is stored in a 4°C refrigerator. This preservation method does not exceed 1 month. The long-term preservation method is to add the Arthrobacter cells to a final concentration of 50% glycerol, mix well, and store in a -80°C refrigerator. This preservation method can reach more than 10 years.

[0053] Example 2: Determination of PE degradation performance by Arthrobacter sp.CY1

[0054] Take the strain stored at -80℃ and streak it on the activation medium plate (LB medium), culture it at 37℃ for 12-24h, pick a single colony of Arthrobacter sp.CY1 strain and inoculate it into LB liquid medium (peptone 10g / L, yeast extract powder 5g / L, sodium chloride 5g / L), and culture it at 28℃ and 150r / min shaking for 8h. 600=0.8-1 fermentation broth as inoculant, the above inoculant was inoculated into an inorganic salt medium (0.70g / LKH2PO4, 0.70g / LK2HPO4, 0.7g / L MgSO4·7H2O, 1.00g / L(NH4)2SO4, 0.005g / LNaCl, 0.002g / LFeSO4·7H2O, 0.002g / LZnSO4·7H2O and 0.001g / LMnSO4·H2O) with an initial PE concentration of 1g / L at an inoculum of 5% (volume fraction) for degradation experiment. 28°C, 150r / min shaking incubator for 90 days, new culture medium was added every 15 days. OD value was detected at its maximum absorbance of 600nm.

[0055] The results of the PE degradation test in the inorganic salt medium are as follows: Figure 5 As shown, the results show that Arthrobacter sp.CY1 strain can significantly degrade PE under liquid culture conditions for a relatively short period of time (30 days), causing the PE film to lose nearly 2.4% of its weight; and can cause the PE film to lose about 3.3% of its weight in 90 days.

[0056] It should be noted that, in this article, the terms "include", "comprises" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, an element defined by the sentence "comprises a ..." does not exclude the existence of other identical elements in the process, method, article or device including the element.

[0057] The serial numbers of the above embodiments of the present invention are only for description and do not represent the advantages or disadvantages of the embodiments.

[0058] 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 preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solutions of the present invention, which should be included in the scope of the claims of the present invention.

Claims

1. An Arthrobacter sp.CY1, characterized in that The Arthrobacter sp.CY1 is deposited in the China General Microbiological Culture Collection Center with the deposit number CGMCC No.32460 and the deposit date October 31, 2024.

2. A bacterial agent, characterized in that Contains the Arthrobacter sp.CY1 described in claim 1.

3. The bacterial agent according to claim 2, characterized in that The dosage form of the bacterial agent includes liquid, powder and granule.

4. The bacterial agent according to claim 2, characterized in that The bacterial agent also includes a carrier, which is at least one of peat, bamboo charcoal powder, kaolin, diatomaceous earth, bentonite and sodium alginate.

5. Use of the Arthrobacter sp.CY1 described in claim 1 or 2 or the bacterial agent described in any one of claims 2 to 4 in degrading plastics.

6. The use according to claim 5, characterized in that: The plastic is polyethylene plastic.

7. A method for degrading PE solution, characterized in that: The following steps are involved: The PE solution to be degraded is mixed and contacted with Arthrobacter sp.CY1 described in claim 1 or the bacterial agent described in claim 3.

8. The method according to claim 7, characterized in that The PE solution also contains KH2PO4, K2HPO4, MgSO4·7H2O, (NH4)2SO4, NaCl, FeSO4·7H2O, ZnSO4·7H2O and MnSO4·H2O.

9. A method for degrading PE film, characterized in that: The method comprises the following steps: inoculating the Arthrobacter sp.CY1 described in claim 1 into an inorganic salt liquid culture medium added with a PE film, and culturing the culture medium at 28° C.-37° C.

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