Pseudomonas stutzeri and application thereof in degradation of PBAT plastic

Abstract

The invention provides pseudomonas stutzeri and application thereof in degradation of PBAT (poly (butylene adipate-co-terephthalate)) plastics, and belongs to the technical field of biology. The enrichment and separation method of the pseudomonas stutzeri comprises the following steps: S1, taking quantitative farmland soil to cover the upper side and the lower side of a PBAT film; s2, taking out the PBAT thin film after the farmland soil is buried for several days, and putting the PBAT thin film into a culture medium containing inorganic salt to be continuously cultured for several days; s3, 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 bacterial suspension after dilution, coating an LB solid culture medium with the bacterial suspension for culture, and then separating out PBAT degrading bacteria; and S4, screening out bacteria which can grow by taking PBAT as a unique carbon source from the separated PBAT degrading bacteria, so as to obtain the pseudomonas stutzeri. The Pseudomonas stutzeri provided by the invention can be used for realizing the biodegradation of PBAT, and the efficiency of degrading PBAT for 15 days by the Pseudomonas stutzeri under the condition that an additional carbon source peptone is added reaches 32.320 + / -4.903%.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to Pseudomonas stutzeri 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 them, 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, and its structure is composed 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. 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 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 deficiencies of the above-mentioned prior art, propose Pseudomonas stutzeri and its application in the degradation of PBAT plastics, screen out a strain of Pseudomonas stutzeri from farmland soil, which promotes the degradation of PBAT, and the efficiency of Pseudomonas stutzeri in degrading PBAT for 15 days reaches 32.320±4.903% under the condition of adding additional carbon source peptone.

[0005] In order to achieve the purpose of the present invention, the following technical scheme is adopted: Pseudomonas stutzeri, whose genome sequence is shown in Sequence Table 1, can survive with PBAT as the only carbon source and can degrade PBAT plastic.

[0006] The classification of Pseudomonas stutzeri of the present invention is named as: Stutzerimonas stutzeri M9-J2 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: 65122 As another technical solution of the present invention, a method for enriching and isolating Pseudomonas stutzeri comprises the following steps: S1. Take a certain amount of farmland soil and cover the upper and lower sides of the PBAT film; S2. After being buried in farmland soil for several days, 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. Among the isolated PBAT-degrading bacteria, bacteria that can grow with PBAT as the sole carbon source are screened out, namely, Pseudomonas stutzeri is obtained.

[0007] Preferably, the enrichment and separation method of Pseudomonas aeruginosa is specifically as follows: Farmland soil was collected from Huizhou. A 5-10 cm farmland 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 farmland 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, Pseudomonas stutzeri, and stored at -80°C. 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 / LKCl 0.2g / L, CaCl2 0.1g / L, FeSO4·2H2O 0.01g / L.

[0008] To achieve the purpose of the present invention, another technical solution of the present invention is the use of the above-mentioned Pseudomonas stutzeri or the Pseudomonas stutzeri prepared by the above-mentioned enrichment and separation method in degrading PBAT plastics. PBAT plastics can be used as the sole carbon source for Pseudomonas stutzeri. When degrading PBAT plastics, Pseudomonas stutzeri can add additional carbon source peptone to improve the degradation efficiency.

[0009] Compared with the prior art, the present invention achieves the following technical effects: The Pseudomonas stutzeri of the present invention has the ability to degrade PBAT; and the Pseudomonas stutzeri of the present invention is isolated from farmland soil, and its source is a microorganism that is isolated to degrade PBAT, and it is highly safe to use. The Pseudomonas stutzeri of the present invention can achieve biological degradation of PBAT. The Pseudomonas stutzeri of the present invention has an efficiency of 32.320±4.903% in degrading PBAT for 15 days under the condition of adding additional carbon source peptone. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] For ease of explanation, the present invention is described in detail with reference to the following specific embodiments and the accompanying drawings.

[0011] Figure 1 The figure is a rod-shaped bacterial morphology of Pseudomonas stutzeri of the present invention observed under a scanning electron microscope.

[0012] Figure 2 For the embodiment of the present invention Stutzerimonas stutzeri Scanning electron microscopy image of M9-J2 colonization on the PBAT surface.

[0013] Figure 3 This is a diagram showing the degradation of PBAT according to an embodiment of the present invention.

[0014] Figure 4 For the embodiment of the present invention Stutzerimonas stutzeri The change in molecular weight of PBAT film after degradation by M9-J2 strain.

[0015] Figure 5 This is a Fourier infrared spectrum diagram of an embodiment of the present invention. DETAILED DESCRIPTION

[0016] 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.

[0017] 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.

[0018] 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.

[0019] Example 1 Stutzerimonas stutzeri Enrichment, separation and identification of M9-J2 1. Enrichment and Isolation of Degrading Strains Farmland soil was collected from Huizhou, and a soil landfill experiment was designed. A 100mg PBAT film was placed flat on a 5-10cm farmland soil sample in a sterile glass container, and then a 5-10cm farmland 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 for 7 days at 150r and 30℃. 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.

[0020] 2. Identification of Degrading Strains (1) Under the scanning electron microscope, the rod-shaped bacteria can be clearly seen ( Figure 1 ).

[0021] (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 M9-J2 sequence had the highest similarity with Stutzerimonas stutzeri DSM 4166, which was 99%. Stutzerimonas stutzeri , named M9-J2. Stutzerimonas stutzeri M9-J2 has a strong ability to degrade PBAT plastic and can survive as the only carbon source of PBAT. Stutzerimonas stutzeri The M9-J2 genome sequence is shown in Sequence Table 1.

[0022] (3) Obtained Pseudomonas stutzeri Stutzerimonas stutzeri Laboratory preservation method of M9-J2: 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.

[0023] Example 2 Stutzerimonas stutzeri M9-J2 colonization ability test on PBAT surface 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 Stutzerimonas stutzeri A single colony of the M9-J2 strain was inoculated into LB liquid medium (peptone 10.0 g; sodium chloride 5.0 g; glucose 1.0 g; yeast extract powder 5.0 g; pH 7.0 ± 0.2; 1000 mL water) and cultured at 30 ° C, 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. SEM images of PBAT after 30 days of culture at 30°C and 150 r / min shaking ( Figure 2 ), Stutzerimonas stutzeri M9-J2 was colonized on the surface of PBAT film.

[0024] Example 3 Stutzerimonas stutzeriStudy on the degradation ability of PBAT by M9-J2 with or without additional carbon source The culture conditions of the M9-J2 strain for degradation of PBAT film: The M9-J2 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, KCl0.2g / 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 on a shaker at 150rpm and 30°C; 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 M9-J2 strain. The results of the PBAT degradation test in the inorganic salt culture medium showed that: Stutzerimonas stutzeri The M9-J2 strain significantly degraded PBAT, and the weight of PBAT could be reduced by nearly 22.316±7.293% in 15 days; Stutzerimonas stutzeri The addition of additional carbon source peptone to the PBAT degradation environment of the M9-J2 strain can improve the strain's ability to degrade PBAT, that is, the PBAT degradation rate increased to 32.320±4.903%. Figure 3 As shown. Stutzerimonas stutzeri The molecular weight changes of PBAT films after degradation by strain M9-J2 are shown in Figure 2. Figure 4 As shown in the figure, the results show that the molecular weight of the degraded PBAT is smaller than that of the PBAT that has not been degraded by the strain. The Fourier infrared spectrum ( Figure 5 )express Stutzerimonas stutzeri The M9-J2 strain degrades the sites that PBAT plastics may attack.

[0025] 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. Pseudomonas stutzeri, It is characterized in that Its genome sequence is shown in Sequence Table 1.

2. The Pseudomonas stutzeri according to claim 1, It is characterized in that Able to survive with PBAT as the only carbon source.

3. The Pseudomonas stutzeri according to claim 1, It is characterized in that Can degrade PBAT plastic.

4. Enrichment and isolation methods of Pseudomonas stutzeri, It is characterized in that The steps include: S1. Take a certain amount of farmland soil and cover the upper and lower sides of the PBAT film; S2. After being buried in farmland soil for several days, 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. Among the isolated PBAT-degrading bacteria, bacteria that can grow with PBAT as the sole carbon source are screened out, namely, Pseudomonas stutzeri is obtained.

5. The method for enriching and isolating Pseudomonas stutzeri according to claim 4, It is characterized in that Specifically: Farmland soil was collected from Huizhou. A 5-10 cm farmland 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 farmland 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 Pseudomonas stutzeri, and stored at -80°C.

6. The method for enriching and isolating Pseudomonas stutzeri according to claim 5, It is characterized in that The formula of liquid culture medium with PBAT as the sole carbon source is: NH 4 Cl 2g / L, KH 2 PO 4 1g / L, Na 2 HPO 4 1.5g / L,MgS 4 O·7H 2 O0.20g / LKCl 0.2g / L, CaCl 2 0.1g / L, FeSO 4 ·2H 2 O 0.01g / L.

7. Use of the Pseudomonas stutzeri described in any one of claims 1 to 3 or the Pseudomonas stutzeri prepared by the enrichment and separation method of any one of claims 4 to 6 in degrading PBAT plastics.

8. The use according to claim 6, It is characterized in that PBAT plastic serves as the sole carbon source for Pseudomonas stutzeri.

9. The use according to claim 6, It is characterized in that Pseudomonas stutzeri added additional carbon source peptone when degrading PBAT plastic.

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