Lysinibacillus capsici and application thereof in degradation of PBAT plastic

Abstract

The invention provides lysinibacillus capsici and application thereof in degradation of PBAT plastic, and belongs to the technical field of biology. Covering the upper side and the lower side of the PBAT film with quantitative beach soil; taking out the PBAT thin film after several days of soil landfill, and putting the PBAT thin film into an inorganic salt-containing culture medium to be continuously cultured for several days; taking a certain amount of the enrichment culture solution, adding sterile normal saline to prepare a bacterial suspension, diluting the bacterial suspension, taking a certain amount of the diluted bacterial suspension, coating an LB solid culture medium with the bacterial suspension for culture, and separating out PBAT degrading bacteria; and screening out bacteria which can grow by taking PBAT as a unique carbon source from the separated PBAT degrading bacteria, thereby obtaining the lysinibacillus capsici. The lysinebacter capsici disclosed by the invention has the capability of degrading PBAT (Poly ( Moreover, the lysinibacillus capsici is separated from beach soil, the source of the lysinibacillus capsici is separated from microorganisms capable of degrading PBAT, and the use safety of the lysinibacillus capsici is high.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to Lysinobacterium capsici and its application in degrading PBAT plastics. Background Art

[0002] As the global plastic pollution problem intensifies, finding sustainable development solutions to replace traditional plastics has become a hot topic in current scientific research and industrial development. In this context, research on biodegradable plastics has attracted much attention, among which polybutadiene adipate terephthalate (PBAT) has become one of the focuses of attention due to its good degradation performance in the environment. PBAT is a polyester plastic whose structure consists of three monomers: terephthalic acid (abbreviated as "T"), adipic acid (abbreviated as "A") and 1,4-butanediol (abbreviated as "B"). Microbial degradation under mild reaction conditions is one of the main ways for PBAT plastic to degrade. Through the action of microorganisms, high molecular polymers can be gradually degraded into smaller molecules, reducing their adverse effects on the ecosystem.

[0003] In natural scenarios such as soil environments, the degradation of PBAT is based on hydrolysis. In soil scenarios, soil quality and humidity are important influencing factors. The differences in the types and numbers of microorganisms affect the degradation rate of PBAT in natural scenarios. Therefore, microbial degradation plays an important role in different environments for PBAT degradation. Therefore, finding a microorganism that can efficiently degrade PBAT has become an important topic of research in environmental protection and sustainable development. Summary of the invention

[0004] The technical problem to be solved by the present invention is that in view of the deficiencies of the above-mentioned prior art, the capsicum lysinophilic bacteria screened out from the beach soil of the present invention promotes the degradation of PBAT, and the capsicum lysinophilic bacteria degrades PBAT with an efficiency of 25.071±0.971% for 15 days under the condition of adding additional carbon source peptone.

[0005] In order to achieve the above object, the present invention adopts the following technical solution: Capsaicinobacterium, whose genome sequence is shown in Sequence Table 1, can survive with PBAT as the sole carbon source and degrade PBAT plastic.

[0006] The classification of the capsicum lysine bacillus of the present invention is named as: Lysinibacillus capsici M32-J1 The name of the depository is: Guangdong Microbiological Culture Collection Center The address of the preservation unit is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou The deposit date is: September 11, 2024 The deposit number is: 65121 Another technical solution of the present invention is a method for enriching and isolating Lysinobacterium capsici, comprising the following steps: S1. Take a certain amount of beach soil and cover the upper and lower sides of the PBAT film; S2. After several days of soil burial, the PBAT film is taken out and placed in a culture medium containing inorganic salts for further cultivation for several days; S3, taking a certain amount of enriched culture fluid, adding sterile physiological saline to prepare a bacterial suspension and diluting it, taking a certain amount of the bacterial suspension after dilution and applying it on LB solid culture medium for culture, and then isolating PBAT-degrading bacteria; S4. The bacteria that can grow with PBAT as the sole carbon source are screened out from the isolated PBAT-degrading bacteria, namely, pepper lysine bacteria are obtained.

[0007] Preferably, the enrichment and separation method is specifically: Beach soil was collected from Huizhou. A 5-10 cm beach soil sample was placed in a sterile glass container and a 100 mg PBAT film was placed flat on top. Then another 5-10 cm beach soil sample was covered on the film to keep the soil moisture content at 60%. After 30 days of soil burial, the PBAT film buried in the soil was taken out. At this time, the film had been partially degraded and enriched with microorganisms. It was placed in a 100 ml conical flask containing 50 ml of inorganic salt culture medium and continued to be cultured for 7 days at 150r and 30°C. Take 1 ml of the enriched culture fluid and add 9 mL of sterile saline to prepare a bacterial suspension. Dilute the prepared bacterial suspension at a certain ratio, take 50 ul of the diluted bacterial suspension and spread it on LB solid culture medium. Observe it every 12 hours. After 2 days, streak culture the single colonies with different morphologies. The isolated single colonies are PBAT-degrading bacteria. The isolated PBAT-degrading bacteria were inoculated into a liquid culture medium with PBAT as the sole carbon source for verification. The inoculation amount was 2%. The liquid culture medium was sterilized at 121°C for 20 min and cultured in a constant temperature shaking incubator at 30°C. After 7 days, the bacteria that could grow with PBAT as the sole carbon source were screened out, namely, Capsicum lysine Bacillus, and stored at -80°C.

[0008] Preferably, the formula of the liquid culture medium with PBAT as the sole carbon source is: NH4Cl 2g / L, KH2PO4 1g / L, Na2HPO4 1.5g / L, MgS4O·7H2O 0.20g / L, KCl 0.2g / L, CaCl2 0.1g / L, FeSO4·2H2O 0.01g / L.

[0009] To achieve the purpose of the present invention, another technical solution of the present invention is: The above-mentioned Capsicum Lysinibacillus or the Capsicum Lysinibacillus prepared by the above-mentioned enrichment and separation method is used to degrade PBAT plastics. PBAT plastics can be used as the sole carbon source for Capsicum Lysinibacillus, and additional carbon source peptone can also be added to improve the efficiency of PBAT degradation.

[0010] Compared with the prior art, the present invention achieves the following technical effects: 1. The capsicum lysine bacillus of the present invention has the ability to degrade PBAT; and the capsicum lysine bacillus of the present invention is isolated from beach soil, and its source is a microorganism that is isolated to degrade PBAT, and it is highly safe to use. The capsicum lysine bacillus of the present invention can achieve biodegradation of PBAT.

[0011] 2. The enrichment and separation method of Lysinobacterium capsici of the present invention is simple, the raw materials used are widely available and easy to obtain, and the preparation efficiency is high.

[0012] 3. When the present invention applies Lysinibacillus capsici to PBAT degradation, the efficiency of Lysinibacillus capsici to PBAT degradation for 15 days reaches 25.071±0.971% under the condition of adding additional carbon source peptone. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] For ease of explanation, the present invention is described in detail with reference to the following specific embodiments and the accompanying drawings.

[0014] Figure 1 This is a graph of the rod-shaped bacteria observed under a scanning electron microscope in Example 1 of the present invention.

[0015] Figure 2 This is a growth curve of Capsicum lysine bacteria under different conditions in Example 1 of the present invention.

[0016] Figure 3 This is a comparison chart of the degradation of PBAT in Example 3 of the present invention when different carbon sources are added.

[0017] Figure 4 Embodiment 3 of the present invention Lysinibacillus capsici The change in molecular weight of PBAT film after degradation by strain M32-J1.

[0018] Figure 5 For Example 4 of the present invention, Lysinibacillus capsici The change in the amount of DMT degradation products after M32-J1 degraded PBAT film. DETAILED DESCRIPTION

[0019] The following are specific embodiments of the present invention and the accompanying drawings to further describe the technical solutions of the present invention, but the present invention is not limited to these embodiments; in the following description, specific details such as specific configurations are provided only to help fully understand the embodiments of the present invention. Therefore, it should be clear to those skilled in the art that various changes and modifications can be made to the embodiments described herein without departing from the scope and spirit of the present invention.

[0020] It should be noted that, in the absence of conflict, the embodiments of the present invention and the features of the embodiments may be combined with each other.

[0021] Unless otherwise specified, the materials, practices and experimental equipment involved in the implementation cases of the present invention are all in line with commercially available products in the relevant chemical and biotechnology fields.

[0022] Example 1 Enrichment, isolation and identification of degradation strains 1. Enrichment and Isolation of Degrading Strains Beach soil was collected from Huizhou, and a soil landfill experiment was designed. A 5-10cm beach soil sample of beach soil was placed flat on a 100mg PBAT film in a sterile glass container, and then a 5-10cm beach soil sample was covered on the film to keep the soil moisture content at 60%. The soil landfill experiment achieved the purpose of enriching PBAT-degrading microorganisms. After 30 days of soil landfill, the PBAT film buried in the soil was taken out. At this time, the film had been partially degraded and enriched with microorganisms. It was placed in a 100ml conical flask containing 50ml of inorganic salt culture medium and continued to be cultured at 150r and 30℃ for 7d. Then 1ml of the enriched culture solution was added to 9mL of sterile saline to prepare a bacterial suspension. The prepared bacterial suspension was diluted in a certain proportion, and 50ul of the diluted bacterial suspension was applied to LB solid culture medium. It was observed every 12h. After 2d, single colonies of different forms were streaked and cultured. The isolated single colonies were PBAT-degrading bacteria. The isolated PBAT degrading bacteria were verified one by one for their ability to degrade PBAT in a liquid culture medium with PBAT as the sole carbon source. The liquid culture medium was sterilized at 121°C for 20 minutes. The formula of the liquid culture medium with PBAT as the sole carbon source was: (NH4Cl 2g / L, KH2PO4 1g / L, Na2HPO4 1.5g / L, MgS4O·7H2O 0.20g / L, KCl 0.2g / L, CaCl2 0.1g / L, FeSO4·2H2O 0.01g / L), cultured in a constant temperature shaker incubator at 30°C, observed after 7 days, and screened out bacteria that can grow with PBAT as the sole carbon source. The purified strain obtained by screening was stored at -80°C.

[0023] 2. Identification of Degrading Strains (1) Under the scanning electron microscope, the rod-shaped bacteria can be clearly seen ( Figure 1 ).

[0024] (2) Molecular identification: The gyrB genome sequence of the isolated strain was analyzed, and the obtained sequence results were compared with the BLAST search results of the National Center for Bioinformation (NCBI) in the United States. It was found that the strain M32-J1 sequence had the highest similarity with Lysinibacillus capsici strain anQ-h6, which was 98%. Lysinibacillus capsici , named M32-J1. Lysinibacillus capsici M32-J1 has a strong ability to degrade PBAT plastics and can survive with PBAT as the only carbon source. When additional nutrients such as peptone are added, the strain's ability to degrade PBAT is improved, and its growth curve is shown in Figure 2. Figure 2 . Lysinibacillus capsici The M32-J1 bacteria have strong vitality and have not been genetically modified, and can effectively degrade PBAT plastic pollutants in the soil. The genome sequence of Lysinibacillus capsici is shown in Sequence Table 1.

[0025] (3) The obtained pepper lysine bacteria ( Lysinibacillus capsici ) Laboratory preservation method of M32-J1: The short-term preservation method is to inoculate the strain into LB liquid culture medium. The LB culture medium formula is: 10.0g peptone; 5.0g sodium chloride; 1.0g glucose; 5.0g yeast extract powder; pH 7.0±0.2; 1000mL water. When the OD600 of the strain is 0.8-1, mix the bacterial liquid with 50% glycerol seed solution in a ratio of 1:1 and store it in a -80℃ refrigerator. This preservation method can last for more than 10 years.

[0026] Example 2 Lysinibacillus capsici M32-J1 Test on PBAT degradation ability Take the strain stored at -80°C and streak it on the activation medium plate (LB medium), incubate it at 30°C for 12-24h, and pick Lysinibacillus capsiciA single colony of the M32-J1 strain was inoculated into LB liquid medium (10.0 g of peptone; 5.0 g of sodium chloride; 1.0 g of glucose; 5.0 g of yeast extract powder; pH 7.0 ± 0.2; 1000 mL of water) and cultured at 30°C and 150 r / min shaking for 6-7 hours. The fermentation broth with a bacterial concentration of O6 D00 = 0.8-1 was used as an inoculant, and the above inoculant was inoculated into an inorganic salt medium (NH4Cl 2g / L, KH2PO4 1g / L, Na2HPO41.5g / L, MgS4O·7H2O 0.20g / L, KCl 0.2g / L, CaCl2 0.1g / L, FeSO4·2H2O 0.01g / L) with an initial PBAT concentration of 2g / L at an inoculum amount of 2% (volume fraction) for degradation experiments. The cells were cultured at 30°C and 150 r / min in a shaking incubator for 15 days, and the degradation of PBAT was tested at 0, 15, and 30 days. The results of the PBAT degradation test in the inorganic salt medium showed that: Lysinibacillus capsici The M32-J1 strain has a significant degradation effect on PBAT, and can reduce the weight of PBAT by nearly 17.366±2.719% in 15 days.

[0027] Example 3 Lysinibacillus capsici M32-J1 Study on the degradation capacity of PBAT under the condition of adding additional carbon source From Example 1, it is concluded that Lysinibacillus capsici M32-J1 has an advantage in degrading PBAT under the condition of adding additional carbon source peptone, so we chose to add additional carbon source peptone and PBAT as the carbon source to the degradation culture medium for the experiment in step 3.

[0028] The culture conditions of the M32-J1 strain for degradation of PBAT film: The M32-J1 strain was inoculated at 2% into a 100ml Erlenmeyer flask containing 50ml of inorganic salt liquid culture medium ((NH4Cl 2g / L, KH2PO4 1g / L, Na2HPO4 1.5g / L, MgS4O·7H2O 0.20g / L, KCl 0.2g / L, CaCl2 0.1g / L, FeSO4·2H2O 0.01g / L)), PBAT film and peptone were added to the culture medium as carbon sources, and cultured at 150rpm and 30°C in a shaker; at the same time, an experimental control group was set up, that is, only PBAT film carbon source was added to the inorganic salt liquid culture medium, and peptone carbon source was not added; the blank control group was only PBAT added to the inorganic salt culture medium, without M32-J1 strain. The degradation rate of PBAT after 15 days of culture showed that: Lysinibacillus capsiciThe addition of additional carbon source peptone to the PBAT degradation environment of the M32-J1 strain can improve the strain's ability to degrade PBAT, that is, the PBAT degradation rate is increased to 25.071±0.971%. Figure 3 As shown. Lysinibacillus capsici The molecular weight changes of PBAT films after degradation by strain M32-J1 are shown in Figure 4 As shown, the results showed that the molecular weight of the degraded PBAT was much smaller than that of the PBAT that was not degraded by the strain.

[0029] Example 4 Lysinibacillus capsici M32-J1 Detection of dimethyl terephthalate (DMT), a degradation product of PBAT The PBAT film samples after degradation in the above experiment were analyzed by GC-MS, equipped with a fused silica capillary column (Elite-5 ms, 30 m × 0.25 mm id × 0.25 μm), and linear temperature gradient analysis was performed (40°C for 1 min, increased to 120°C at a rate of 15°C / min, maintained for 2 min, and then increased to 300°C at a rate of 10°C / min, maintained for 5 min). The injection temperature was set to 250°C. The mass spectrum was obtained by electron impact ionization at 70 eV, and the scanning spectrum was obtained in the range of 45 ~ 450m / z. The peak time of the DMT product is around 6.35min. By Lysinibacillus capsici The changes in the amount of DMT degradation products after M32-J1 degradation of PBAT film (e.g. Figure 5 ), it can be seen Lysinibacillus capsici The action site of M32-J1 in degrading PBAT plastic polymers is the ester bond.

[0030] Those skilled in the art to which the present application belongs may make various modifications or additions to the specific embodiments described or replace them in a similar manner, but they will not deviate from the inventive concept of the present application or exceed the scope defined by the attached claims.

Claims

1. Capsicum lysinic acid bacteria, characterized in that Its genome sequence is shown in Sequence Table 1.

2. The Lysinibacillus capsici according to claim 1, characterized in that Able to survive with PBAT as the only carbon source.

3. The Lysinibacillus capsici according to claim 1, characterized in that Can degrade PBAT plastic.

4. A method for enriching and isolating Lysinibacterium capsici, characterized in that: The steps include: S1. Take a certain amount of beach soil and cover the upper and lower sides of the PBAT film; S2. After several days of soil burial, the PBAT film is taken out and placed in a culture medium containing inorganic salts for further cultivation for several days; S3, taking a certain amount of enriched culture fluid, adding sterile physiological saline to prepare a bacterial suspension and diluting it, taking a certain amount of the bacterial suspension after dilution and applying it on LB solid culture medium for culture, and then isolating PBAT-degrading bacteria; S4. The bacteria that can grow with PBAT as the sole carbon source are screened out from the isolated PBAT-degrading bacteria, namely, pepper lysine bacteria are obtained.

5. The method for enriching and separating Lysinibacillus capsici according to claim 4, characterized in that: Specifically: Beach soil was collected from Huizhou. A 5-10 cm beach soil sample was placed in a sterile glass container and a 100 mg PBAT film was placed flat on top. Then another 5-10 cm beach soil sample was covered on the film to keep the soil moisture content at 60%. After 30 days of soil burial, the PBAT film buried in the soil was taken out. At this time, the film had been partially degraded and enriched with microorganisms. It was placed in a 100 ml conical flask containing 50 ml of inorganic salt culture medium and continued to be cultured for 7 days at 150r and 30°C. Take 1 ml of the enriched culture fluid and add 9 mL of sterile saline to prepare a bacterial suspension. Dilute the prepared bacterial suspension at a certain ratio, take 50 ul of the diluted bacterial suspension and spread it on LB solid culture medium. Observe it every 12 hours. After 2 days, streak culture the single colonies with different morphologies. The isolated single colonies are PBAT-degrading bacteria. The isolated PBAT-degrading bacteria were inoculated into a liquid culture medium with PBAT as the sole carbon source for verification. The inoculation amount was 2%. The liquid culture medium was sterilized at 121°C for 20 min and cultured in a constant temperature shaking incubator at 30°C. After 7 days, the bacteria that could grow with PBAT as the sole carbon source were screened out, namely, Capsicum lysine Bacillus, and stored at -80°C.

6. The method for enriching and isolating Lysinibacillus capsici according to claim 5, characterized in that: The formula of liquid culture medium with PBAT as the sole carbon source is: NH4Cl 2g / L, KH2PO4 1g / L, Na2HPO41.5g / L, MgS4O·7H2O0.20g / L, KCl 0.2g / L, CaCl2 0.1g / L, FeSO4·2H2O 0.01g / L.

7. Use of the capsicum lysine bacillus described in any one of claims 1 to 3 or the capsicum lysine bacillus prepared by the enrichment and separation method of any one of claims 4 to 5 in degrading PBAT plastics.

8. The use according to claim 6, characterized in that: PBAT plastic serves as the sole carbon source for Lysinobacterium capsici.

9. The use according to claim 6, characterized in that: Capsicum lysine bacteria added additional carbon source peptone when degrading PBAT plastic.

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