Bacillus proteolyticus and application thereof

Abstract

Bacillus proteolyticus and application thereof, the present application relates to a kind of bacteria and its application.The present application provides a kind of bacillus proteolyticus and its application, the strain is a kind of bacteria with degradation PBAT mulch microplastics.The bacillus proteolyticus L6 of the present application is preserved in China Microbial Culture Collection Center, and the preservation number is CGMCC No.26901.The bacillus proteolyticus L6 of the present application can efficiently degrade PBAT mulch microplastics.The strain described in the present application lays a good foundation for the development and utilization of degradable mulch microplastics.

Description

Technical Field

[0001] This invention relates to a bacterium and its applications. Background Technology

[0002] Mulch film, with its functions of warming, water retention, weed and pest control, and yield enhancement, is widely used in agricultural production. Due to difficulties in recycling, mulch film plastic breaks down into microplastics under the influence of tillage, ultraviolet radiation, and biodegradation. Microplastics refer to plastic fragments or particles with a diameter of less than 5 mm. The main microplastics from broken mulch film in soil are polybutylene adipate / terephthalate (PBAT). Mulch film, as an in-situ source of microplastics, is a significant source of microplastics in farmland soil, and the resulting microplastic pollution problem cannot be ignored, posing a major threat to the agricultural ecosystem. Continuous mulching can lead to directed mutations in soil microorganisms, preserving mutated genes capable of degrading microplastics and producing microplastic-degrading bacteria. Therefore, by screening degrading bacteria and identifying their degradation capabilities, strains capable of efficiently degrading mulch film microplastics can be obtained, providing a theoretical basis and technical support for the prevention and control of microplastic pollution in farmland soil. This is of great significance for mitigating and controlling pollution caused by agricultural film microplastics and improving soil health and the ecological environment. Summary of the Invention

[0003] This invention provides a strain of Bacillus pyrenoidosa and its application, wherein the strain is a bacterium capable of degrading PBAT mulch film microplastics.

[0004] The present invention relates to Bacillus proteolyticus L6, which is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 26901.

[0005] The application of Bacillus proteolyticus L6 described in this invention in the degradation of PBAT mulch film microplastics.

[0006] The Bacillus proteolyticus L6 strain described in this invention was used for laboratory simulation, and OD values ​​were measured before and after degradation. 600nm Scanning electron microscopy revealed that the PBAT microplastics became rough and uneven, exhibiting obvious erosion pits and clear cracks. Fourier transform infrared spectroscopy showed that at 3432 cm⁻¹... -1 (3500-3100cm -1 The additional absorption peak at region (2930 cm⁻¹) can be attributed to -OH, indicating that after Bacillus proteolyticus L6 acts on the plastic, it introduces polar functional groups, increasing the structural instability and causing degradation. -1 2850cm-1 This corresponds to a characteristic peak of the methylene group, observed in L6-treated plastic at 2925 cm⁻¹. -1 The peak shifted (+2cm) at 1761cm. -1 The absorption peak of the tensile vibration at C=O has shifted to 1757 cm⁻¹. -1 1593cm -1 The displacement at this point is due to the vibration of the benzene ring skeleton (Vc = c), reaching 1596 cm. -1 1137cm -1 The absorption peaks of the bending vibration and CO stretching vibration in the CH plane of the benzene ring shifted to 1135 cm⁻¹. -1 Furthermore, at 1461cm -1 and 528cm -1 There is an additional absorption peak at 1461 cm⁻¹ -1 The bending vibration within the CH plane indicates that inoculation with the *Bacillus pyrenoidosa* L6 of this invention can biodegrade PBAT, leading to changes in the chemical functional groups on its surface. The addition of *Bacillus pyrenoidosa* L6 to PBAT microplastics resulted in a degradation rate of 25.4%, demonstrating that *Bacillus pyrenoidosa* L6 has a good degradation effect on PBAT mulch film microplastics.

[0007] The *Bacillus proteolyticus* L6 strain of this invention can efficiently degrade PBAT mulch film microplastics under shaking culture conditions at 28°C. The strain described in this invention lays a solid foundation for the development and utilization of biodegradable mulch film microplastics.

[0008] The Bacillus proteolyticus L6 strain of this invention is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, on March 24, 2023. Attached Figure Description

[0009] Figure 1 The colony morphology of Bacillus proteolyticus L6 on LB medium;

[0010] Figure 2 Phylogenetic tree of Bacillus proteolyticus L6;

[0011] Figure 3 The degradation rate of microplastics after Bacillus proteolyticus L6 was re-inoculated into inorganic salt culture medium;

[0012] Figure 4The microstructure of microplastics after Bacillus proteolyticus L6 was re-inoculated with inorganic salt culture medium;

[0013] Figure 5 The variation of ΔOD600 in Bacillus proteolyticus L6 with PBAT mulch film microplastics as the sole carbon source;

[0014] Figure 6 This is a schematic diagram showing Bacillus proteolyticus L6 attached to the surface of microplastics as observed under a scanning electron microscope.

[0015] Figure 7 The changes in the chemical functional groups of microplastics in Bacillus proteolyticus L6 after inoculation with inorganic salt culture medium. Detailed Implementation

[0016] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0017] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other.

[0018] Specific implementation method one: Bacillus proteolyticus L6 in this implementation method is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, on March 24, 2023, with accession number CGMCC No. 26901.

[0019] The method for obtaining Bacillus proteolyticus L6 in this embodiment is as follows:

[0020] I. Isolation and purification of bacteria: In 2021, 1g of soil from a peanut field in Fuyu City, Jilin Province, with a 10-year history of plastic film mulching, was weighed and added to a test tube containing 9mL of liquid micro-carbon source culture medium. After shaking at 180 rpm in a shaker at 28°C for 30 min, dilute sequentially to 10⁻⁶. -4Concentration was used for the solid plate enrichment of microplastic-degrading bacteria. Because some soil microorganisms have poor resistance and cannot quickly adapt to an environment where plastic is the sole carbon source, trace carbon sources were added to the first enrichment medium to improve the survival rate of various microorganisms. Sterilized 2.5×2.5cm plastic squares of different sizes were evenly spread on a solid trace carbon source medium, and 100μL of a 10% concentration was taken. -4 Soil suspensions were spread onto solid culture media and incubated at 28°C for 10 days. A blank control was prepared by incubating a plastic film-solid microcarbon source medium without soil suspension on the same medium under the same conditions. After 10 days, the film was transferred to a new solid microcarbon source medium for further enrichment for another 10 days. Solid plate enrichment culture: After 20 days of plate enrichment, 2.5 × 2.5 cm microplastic films on the plates were washed with sterile 0.1 M PBS (pH 7.2) with shaking to prepare a bacterial suspension. 100 μL of the bacterial suspension was spread onto an inorganic salt medium lined with a 9 cm diameter plastic film and incubated at 28°C for 30 days. A blank control was prepared by incubating a microplastic film-solid inorganic salt medium without bacterial suspension on the same conditions. After 30 days of solid plate culture, visible colonies were picked and streaked onto LB agar for purification to isolate bacteria with potential degradation capabilities.

[0021] II. Identification of Microplastic Degrading Bacteria: DNA was extracted from the strain isolated in step one using an alkaline lysis method. 16S rDNA amplification was performed using primers 27F / 1492R and P1 / P6, followed by sequencing. Sequence alignment was performed using the Ezbiocloud database, showing 100% similarity to (MK567783.1) Bacillus proteolyticus I-16-d. A phylogenetic tree was constructed using MEGA 7.0. Strain L6 was ultimately determined to belong to the same branch as Bacillus proteolyticus, with 100% similarity. Based on morphological identification, it was finally named Bacillus proteolyticus L6.

[0022] The specific morphological characteristics and molecular identification methods of Bacillus proteolyticus L6 are as follows:

[0023] (1) In this embodiment, the Bacillus proteolyticus L6 colonies are milky yellow, smooth and transparent, round with neat edges, and their colony morphology on LB medium is as follows. Figure 1 As shown;

[0024] (2) Molecular identification: 16S rRNA gene sequence and phylogenetic analysis of bacterial L6

[0025] The 16S rRNA gene sequence and DNA sequence of strain L6 were obtained, as shown in SEQ ID NO: 1. BLAST alignment analysis was then performed with known sequences from the Ezbiocloud database. A phylogenetic tree was constructed using MEGA 7.0, confirming that strain L6 belongs to the same branch as *Bacillus proteolyticus*, with 100% similarity. Based on morphological characteristics and molecular biological identification, strain L6 is confirmed to be *Bacillus proteolyticus*. The phylogenetic tree of *Bacillus proteolyticus* L6 is shown below. Figure 2 As shown.

[0026] Specific Implementation Method Two: The application of Bacillus proteolyticus L6 as described in Specific Implementation Method One in the degradation of PBAT mulch film microplastics.

[0027] Specific application methods:

[0028] I. Treatment of PBAT plastic film: On a sterile workbench, cut the PBAT plastic film into 5×5mm fragments, weigh them accurately, and place them in sterile 10mL centrifuge tubes for sterile treatment. Soak them in 2% SDS, 75% ethanol, and 95% ethanol for 4 hours or more respectively. During this period, use a CNC ultrasonic cleaner and a vortex mixer to clean them for 30 minutes each. After soaking, rinse them 3 times with sterile water. After absorbing the moisture adhering to the surface of the plastic film with sterile filter paper on a clean bench, sterilize them with ultraviolet light for 15 minutes.

[0029] 2. After culturing the obtained Bacillus proteolyticus L6 in LB broth at 28℃ for 18 h, the bacterial cells were obtained by centrifugation at 12,000 rpm for 10 min. The cells were resuspended in an equal volume of PBS and centrifuged twice. The obtained bacterial cells were then prepared into 1×10⁻⁶ saturates using PBS. 7 bacterial suspension with cfu / mL;

[0030] 3. Add 0.1g (0.35%, w / v) of the PBAT plastic film treated in step 1 and 3ml of bacterial solution (10%, v / v) from step 2 to 30mL of inorganic salt liquid culture medium, and set up a negative control group (CK) with only sterile water added. OD was measured after 11 weeks of degradation. 600nm ;

[0031] IV. Weigh the results using an electronic balance, observe the degradation of the plastic using a scanning electron microscope, and determine the changes in the chemical functional groups of the PBAT mulch film microplastics using Fourier transform infrared spectroscopy.

[0032] The experimental results show that: Figure 3 The microplastic degradation rate of strain L6 after inoculation with inorganic salt medium; Figure 4 The microscopic morphology of microplastics in Bacillus proteolyticus L6 after inoculation with inorganic salt culture medium was shown. PBAT-CK served as the negative control group, and L6 was the L6 treatment group in this example. Figure 3 and Figure 4 It can be seen that in the treatment with the addition of Bacillus proteolyticus L6 of the present invention, the strain was observed to adhere to the surface of PBAT mulch film microplastics, and the surface roughness of the microplastics increased, with local unevenness and many obvious pits and grooves. It can significantly degrade PBAT mulch film microplastics, with a degradation rate of 25.4%, while there was no significant change in PBAT mulch film microplastics in PBAT-CK (negative control group). This indicates that Bacillus proteolyticus L6 of the present invention has a degradation effect on PBAT mulch film microplastics.

[0033] Figure 5 To investigate the ΔOD of Bacillus proteolyticus L6 strain in PBAT mulch film microplastics as the sole carbon source. 600 Change; from Figure 5 It can be seen that strain L6 of the present invention has the ability to degrade PBAT mulch film microplastics and continues to grow using PBAT as a carbon source for 11 weeks.

[0034] Figure 6 This is a schematic diagram of Bacillus proteolyticus L6 attached to the surface of microplastics as observed under a scanning electron microscope. The scanning electron microscope shows that the PBAT microplastics become rough and uneven, with obvious erosion holes and clear cracks and other micromorphological features. Figure 7 The changes in the chemical functional groups of microplastics in Bacillus proteolyticus L6 after re-inoculation with inorganic salt culture medium were analyzed by Fourier transform infrared spectroscopy, showing changes at 3432 cm⁻¹. -1 (3500-3100cm -1 The additional absorption peak at region (2930 cm⁻¹) can be attributed to -OH, indicating that after Bacillus proteolyticus L6 acts on the plastic, it introduces polar functional groups, increasing the structural instability and causing degradation. -1 2850cm -1 This is a characteristic peak of the methylene group, observed at 2925 cm⁻¹ in plastic treated with Bacillus proteolyticus L6. -1The peak shifted (+2cm) at 1761cm. -1 The absorption peak of the tensile vibration at C=O has shifted to 1757 cm⁻¹. -1 1593cm -1 The displacement at this point is due to the vibration of the benzene ring skeleton (Vc = c), reaching 1596 cm. -1 1137cm -1 The absorption peaks of the bending vibration and CO stretching vibration in the CH plane of the benzene ring shifted to 1135 cm⁻¹. -1 Furthermore, at 1461cm -1 and 528cm -1 There is an additional absorption peak at 1461 cm⁻¹ -1 This represents bending vibrations within the CH plane. This indicates that the *Bacillus proteoglycans* L6 strain of this invention biodegrades PBAT, leading to changes in the chemical functional groups on its surface.

[0035] The *Bacillus proteolyticus* L6 strain of this invention can efficiently degrade PBAT mulch film microplastics under shaking culture conditions at 28°C. The strain described in this invention lays a solid foundation for the development and utilization of biodegradable mulch film microplastics.

Claims

1. A strain of Bacillus proteinophilus ( Bacillus proteolyticus L6, characterized in that, The protein-degrading Bacillus ( Bacillus proteolyticus L6 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 26901.

2. The *Bacillus proteolyticus* as described in claim 1 (… Bacillus proteolyticus Application of L6 in degradable PBAT mulch film microplastics.

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