Lysinibacillus peptoniphilus and its application in degrading PBAT plastic

Abstract

The application provides a lysine bacillus capsici and application thereof in degrading PBAT plastic, and belongs to the technical field of biotechnology. A certain amount of sandy soil is covered on the upper and lower sides of a PBAT film; the PBAT film is taken out after being filled with soil for several days, and is placed in an inorganic salt culture medium for further culture for several days; a certain amount of the enrichment culture liquid is added into sterile normal saline to prepare a bacterial suspension and dilution; a certain amount of the bacterial suspension after dilution is spread on an LB solid culture medium for culture, and a PBAT degrading bacterium is isolated; and the PBAT degrading bacterium is screened from the isolated PBAT degrading bacterium, so that the lysine bacillus capsici is obtained. The lysine bacillus capsici has the degradation capacity for PBAT; and the lysine bacillus capsici is isolated from sandy soil, is derived from the microorganism isolated for degrading PBAT, and has high safety in use.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to *Bacillus capsicisae* and its application in the degradation of PBAT plastics. Background Technology

[0002] With the escalating global problem of plastic pollution, finding sustainable development solutions to replace traditional plastics has become a hot topic in current scientific research and industrial development. Against this backdrop, research on biodegradable plastics has attracted considerable attention, with polybutadiene terephthalate (PBAT) being a focus due to its excellent degradation performance in the environment. PBAT is a polyester plastic composed of three monomers: terephthalic acid (T), adipic acid (A), and 1,4-butanediol (B). Mild microbial degradation is one of the main pathways for PBAT plastic degradation. Through the action of microorganisms, the high molecular weight polymer can gradually degrade into smaller molecules, mitigating its adverse impact on the ecosystem.

[0003] In natural environments such as soil, PBAT degradation is based on hydrolysis. In soil environments, soil type and moisture are important influencing factors, and the differences in the types and quantities of microorganisms affect the degradation rate of PBAT in natural environments. Therefore, microbial degradation plays an important role in the different environments in which PBAT degrades. Thus, finding a microorganism capable of efficiently degrading PBAT has become an important research topic in environmental protection and sustainable development. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to address the shortcomings of the prior art. The present invention promotes the degradation of PBAT by the capsicum lysinophile screened from beach soil, and the capsicum lysinophile achieves a degradation efficiency of 25.071±0.971% for PBAT after 15 days when additional carbon source peptone is added.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] The genome sequence of *Lysinobacillus capsici* is shown in Sequence Listing 1. It can survive using PBAT as the sole carbon source and degrade PBAT plastic.

[0007] The classification name of the *Lysinobacter capsici* of this invention is: Lysinibacillus capsici M32-J1

[0008] The name of the depository is: Guangdong Provincial Center for the Preservation of Microbial Cultures

[0009] The address of the depository is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0010] The preservation date is September 11, 2024.

[0011] The accession number is: 65121

[0012] Another technical solution of the present invention is a method for enriching and isolating *Lysinobacter capsici*, comprising the following steps:

[0013] S1. Take a certain amount of sandy soil and cover the top and bottom sides of the PBAT film;

[0014] S2. After being buried in soil for several days, the PBAT film was removed and placed in a culture medium containing inorganic salts for further cultivation for several days.

[0015] S3. Take a certain amount of enriched culture medium, add sterile physiological saline to prepare a bacterial suspension and dilute it. After dilution, take a quantitative amount of the bacterial suspension and spread it on LB solid medium for culture. Then isolate the PBAT degrading bacteria.

[0016] S4. Among the isolated PBAT-degrading bacteria, bacteria that can grow using PBAT as the sole carbon source are screened out, thus obtaining *Bacillus capsicisae*.

[0017] The preferred enrichment and separation method is as follows:

[0018] Beach soil was collected from Huizhou. A 5-10cm layer of beach soil was placed in a sterile glass container, and a 100mg PBAT film was placed on top. Then, another 5-10cm layer of beach soil was placed on top of the film, maintaining the soil moisture content at 60%.

[0019] Thirty days after the soil was buried, the PBAT film buried in the soil was removed. At this time, the film had been partially degraded and enriched with microorganisms. It was then placed in a 100ml Erlenmeyer flask containing 50ml of inorganic salt culture medium and cultured for another 7 days at 150 rpm and 30℃.

[0020] Take 1 ml of enrichment culture medium and add 9 mL of sterile physiological saline to prepare a bacterial suspension. Dilute the prepared bacterial suspension according to a certain ratio. Take 50 μL of the diluted bacterial suspension and spread it on LB solid medium. Observe once every 12 h. After 2 days, streak culture of single colonies of different morphologies. The isolated single colonies are PBAT degrading bacteria.

[0021] 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 shaker at 30°C. After 7 days, the bacteria that could grow with PBAT as the sole carbon source were screened out, which is the capsicum lysinophil. The bacteria were then stored at -80°C.

[0022] The preferred formulation of the liquid culture medium using PBAT as the sole carbon source is as follows: 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.

[0023] To achieve the objective of this invention, another technical solution is as follows:

[0024] In the application of the above-mentioned *Bacillus capsicisae* or the *Bacillus capsicisae* prepared by the above-mentioned enrichment and separation methods in the degradation of PBAT plastic, PBAT plastic can be used as the sole carbon source for *Bacillus capsicisae*, or additional carbon source peptone can be added to improve the efficiency of PBAT degradation.

[0025] Compared with the prior art, the present invention achieves the following technical effects:

[0026] 1. The *Capsicum lysinophil* of the present invention has the ability to degrade PBAT; moreover, the *Capsicum lysinophil* of the present invention is isolated from sandy soil, its source is a microorganism isolated to degrade PBAT, and its use is highly safe. The biodegradation of PBAT can be achieved using the *Capsicum lysinophil* of the present invention.

[0027] 2. The enrichment and separation method of the capsicum lysinophil in this invention is simple, the raw materials used are widely available and easy to obtain, and the preparation efficiency is high.

[0028] 3. When the present invention applies Capsicum lysinophila to the degradation of PBAT, the efficiency of Capsicum lysinophila in the degradation of PBAT after 15 days is 25.071±0.971% under the condition of adding additional carbon source peptone. Attached Figure Description

[0029] For ease of explanation, the present invention will be described in detail below with reference to specific embodiments and accompanying drawings.

[0030] Figure 1 This is a scanning electron microscope image of the rod-shaped bacteria *Lysinobacillus capsici* as observed in Example 1 of the present invention.

[0031] Figure 2 This is a growth curve diagram of *Lysinobacter capsici* under different conditions in Example 1 of the present invention.

[0032] Figure 3 This is a comparison chart of the degradation of PBAT under different carbon source conditions in Example 3 of the present invention.

[0033] Figure 4 This is Embodiment 3 of the present invention. Lysinibacillus capsiciThe graph shows the change in molecular weight of the PBAT film after degradation by strain M32-J1.

[0034] Figure 5 This is for embodiment 4 of the present invention. Lysinibacillus capsici Graph showing the change in the amount of DMT degradation products after M32-J1 degrades PBAT film. Detailed Implementation

[0035] The following are specific embodiments of the present invention, described in conjunction with the accompanying drawings, to further illustrate the technical solutions of the present invention. However, the present invention is not limited to these embodiments. Specific details, such as particular configurations, are provided in the following description merely to aid in a comprehensive understanding of the embodiments of the present invention. Therefore, those skilled in the art should understand that various changes and modifications can be made to the embodiments described herein without departing from the scope and spirit of the present invention.

[0036] It should be noted that, unless otherwise specified, the embodiments and features described in this invention can be combined with each other.

[0037] Unless otherwise specified, the materials, practices, and experimental equipment involved in the embodiments of this invention are all commercially available products in the relevant chemical and biotechnology fields.

[0038] Example 1: Enrichment, Isolation and Identification of Degrading Strains

[0039] 1. Enrichment and isolation of degrading strains

[0040] Sandy soil samples were collected from Huizhou. A soil burial experiment was designed, in which a 5-10cm layer of sandy soil was placed in a sterile glass container, and a 100mg PBAT film was placed flat on top. Another 5-10cm layer of sandy soil was then placed on top of the film, maintaining a soil moisture content of 60%. The purpose of the soil burial experiment was to enrich PBAT-degrading microorganisms. After 30 days of soil burial, the PBAT film was removed. At this point, the film had been partially degraded and enriched with microorganisms. It was then placed in a 100ml Erlenmeyer flask containing 50ml of inorganic salt medium and cultured for 7 days at 150rpm and 30℃. Then, 1ml of the enriched culture was added to 9mL of sterile physiological saline to prepare a bacterial suspension. The prepared bacterial suspension was diluted according to a certain ratio, and 50ul of the diluted suspension was spread on LB solid medium. Observations were made every 12 hours. After 2 days, single colonies of different morphologies were streaked, and the isolated single colonies were identified as PBAT-degrading bacteria. The ability of isolated PBAT-degrading bacteria to degrade PBAT was verified one by one in a liquid medium with PBAT as the sole carbon source. The liquid medium was sterilized at 121°C for 20 min. The formulation of the liquid 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). The medium was incubated in a constant temperature shaker at 30°C for 7 days. After observation, bacteria that could grow with PBAT as the sole carbon source were screened out. The purified strain obtained by screening was stored at -80°C.

[0041] 2. Identification of degrading strains

[0042] (1) Under a scanning electron microscope, the morphology of rod-shaped bacteria can be clearly observed. Figure 1 ).

[0043] (2) Molecular identification: The genome sequence of the isolated strain was analyzed. A BLAST search and comparison of the sequence results with the National Center for Bioinformatics (NCBI) revealed that the sequence of strain M32-J1 had the highest similarity (98%) to Lysinibacillus capsici strain anQ-h6. Therefore, the isolated strain was identified as... Lysinibacillus capsici It was named M32-J1. The above strain... Lysinibacillus capsici M32-J1 exhibits strong degradation ability against PBAT plastics and can survive using PBAT as its sole carbon source. Furthermore, the degradation ability of this strain against PBAT is enhanced when peptone is added as an additional nutrient. Its growth curve is shown in [Figure number missing]. Figure 2 . Lysinibacillus capsiciThe M32-J1 bacteria are highly vigorous, have not undergone genetic modification, and can effectively degrade PBAT plastic pollutants in soil. The genome sequence of *Lysinobacillus capsici* is shown in Sequence Listing 1.

[0044] (3) The obtained capsicum lysinophil ( Lysinibacillus capsici Laboratory preservation method for M32-J1: For short-term preservation, the strain is inoculated into LB liquid medium. The LB medium formula is: 10.0g peptone; 5.0g sodium chloride; 1.0g glucose; 5.0g yeast extract; pH 7.0±0.2; 1000mL water. When the OD600 of the strain is 0.8-1, the bacterial culture is mixed with 50% glycerol preservation solution at a ratio of 1:1 and then stored in a -80℃ freezer. This preservation method can achieve a preservation period of more than 10 years.

[0045] Example 2 Lysinibacillus capsici Test of M32-J1's ability to degrade PBAT

[0046] Streak the bacterial strain stored at -80°C onto an activation medium plate (LB medium), incubate at 30°C for 12-24 hours, and then pick up the culture. Lysinibacillus capsici Single colonies of strain M32-J1 were inoculated into LB liquid medium (10.0 g peptone; 5.0 g sodium chloride; 1.0 g glucose; 5.0 g yeast extract; pH 7.0 ± 0.2; 1000 mL water) and cultured at 30°C and 150 rpm for 6-7 h on a shaker. Fermentation broth with a cell concentration of O6 D00 = 0.8-1 was used as inoculum. This inoculum was inoculated at a rate of 2% (v / L) into an inorganic salt medium with an initial PBAT concentration of 2 g / L (NH4Cl 2 g / L, KH2PO4 1 g / L, Na2HPO4 1.5 g / L, MgS4O·7H2O 0.20 g / L, KCl 0.2 g / L, CaCl2 0.1 g / L, FeSO4·2H2O 0.01 g / L) for degradation experiments. The PBAT was cultured at 30°C and 150 rpm for 15 days, and the degradation was assessed at 0, 15, and 30 days. The results of the PBAT degradation assay in the inorganic salt medium showed that: Lysinibacillus capsici The M32-J1 strain significantly degrades PBAT, reducing its weight by approximately 17.366 ± 2.719% in 15 days.

[0047] Example 3 Lysinibacillus capsici Investigation of the PBAT degradation ability of M32-J1 under the condition of adding additional carbon source

[0048] From Example 1 Lysinibacillus capsiciM32-J1 showed an advantage in degrading PBAT under the condition of adding extra carbon source peptone, so the degradation medium with added extra carbon source peptone and PBAT as carbon source was selected for the experiment in step 3.

[0049] Culture conditions for PBAT membrane degradation by strain M32-J1: Strain M32-J1 was inoculated at a 2% inoculum into 100ml Erlenmeyer flasks containing 50ml of inorganic salt liquid 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 membrane and peptone were added as carbon sources. The culture was carried out on a shaker at 150rpm and 30°C. A control group was also set up, with inorganic salt liquid medium containing only PBAT membrane as carbon source and no peptone. A blank control group was formed with inorganic salt medium containing only PBAT and no strain M32-J1. The degradation rate of PBAT after 15 days of culture showed that: Lysinibacillus capsici Adding extra carbon source peptone to the environment in which PBAT is degraded enhances the PBAT degradation ability of strain M32-J1, increasing the PBAT degradation rate to 25.071 ± 0.971%. The PBAT degradation process is as follows: Figure 3 As shown. At the same time Lysinibacillus capsici The change in molecular weight of the PBAT film after degradation by strain M32-J1 is as follows: Figure 4 As shown, the results indicate that the molecular weight of the degraded PBAT is much smaller than that of the undegraded PBAT.

[0050] Example 4 Lysinibacillus capsici Detection of dimethyl terephthalate (DMT), a degradation product of PBAT, using M32-J1

[0051] The PBAT film samples after degradation in the above experiments were analyzed by GC-MS using a fused silica capillary column (Elite-5 ms, 30 m × 0.25 mm id × 0.25 μm). Linear temperature gradient analysis was performed (40°C for 1 min, increased to 120°C at a rate of 15°C / min, held for 2 min, then increased to 300°C at a rate of 10°C / min, held for 5 min). The injection inlet temperature was set to 250°C. Mass spectra were obtained by electron shock ionization at 70 eV, and scans were obtained in the range of 45–450 m / z. The elution time of the DMT product was approximately 6.35 min. Lysinibacillus capsiciChanges in the amount of DMT degradation products after M32-J1 degradation of PBAT film (e.g.) Figure 5 ), we can know Lysinibacillus capsici The active site of M32-J1 in degrading PBAT plastic polymers is at the ester bond.

[0052] Those skilled in the art to which this application pertains may make various modifications or additions to the specific embodiments described, or adopt similar methods to replace them, without departing from the inventive concept of this application or exceeding the scope defined by the appended claims.

Claims

1. Capsicum Lysine Bacillus ( Lysinibacillus capsici M32-J1, characterized in that, Its biological preservation number is CDMCC No: 65121, and the preservation institution is Guangdong Provincial Center for Microbial Culture Collection.

2. The *Lysinobacillus capsici* according to claim 1, characterized in that, This includes sequences such as those shown in Sequence 1.

3. The *Lysinobacillus capsici* according to claim 1, characterized in that, It can survive using PBAT as its sole carbon source.

4. The *Lysinobacillus capsici* according to claim 1, characterized in that, It can degrade PBAT plastic.

5. The application of the capsicum lysinophilus according to any one of claims 1-4 in the degradation of PBAT plastic.

6. The application according to claim 5, characterized in that, PBAT plastic serves as the sole carbon source for *Lysinobacter capsici*.

7. The application according to claim 6, characterized in that, Capsicum lysinophilus adds extra carbon source peptone when degrading PBAT plastic.

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