Aeromonas sp. and application thereof in degrading polyethylene plastic

Abstract

The application provides an Aquabacterium sp. A3 strain collected from Haiong Island in Guangzhou City, Guangdong Province, and obtained through artificial enrichment culture and separation and purification. Quantitative analysis is performed by using a gas chromatograph (GC), and it is found that the strain Aquabacterium sp. A3 has the ability of mineralizing PE and can mineralize PE into CO2. Surface functional group analysis of plastic is performed by using a Fourier infrared spectrometer (FTIR-ATR mode), and it is found that the addition of the strain Aquabacterium sp. A3 of the application obviously changes the content and quantity of the functional groups on the surface of PE microplastic, which proves that the strain has the ability of efficiently mineralizing the surface of PE microplastic and has the potential of being applied to the environment such as groundwater to realize biodegradation of microplastic.

Description

Technical Field

[0001] This invention relates to the field of microbiology, and in particular to a strain of Aquabacterium and its application in the degradation of polyethylene plastics. Background Technology

[0002] Plastic is an indispensable part of the human material world. With its continuous use in production and daily life, plastic waste is generated in the environment. Because plastic is difficult to degrade naturally, it accumulates in the environment, and environmental problems such as "white pollution" caused by plastic pollution have become a global challenge. Currently, simple physicochemical methods are insufficient to completely remove plastic; they can only further refine existing plastic pollutants into microplastics. Microplastics are hydrophobic, small in size, and have a large specific surface area, making them prone to combining with other water pollutants to form complex pollutants, which then migrate continuously in nature, increasing the difficulty of environmental pollution control. Biodegradation is one of the potential means to control plastic pollutants. Microorganisms can completely mineralize plastic components into CO2 through metabolism, thereby removing plastic waste residues in the environment. Polyethylene (PE) plastic, the most common type of plastic on the market, is relatively difficult to degrade. Therefore, analyzing the functional microbial populations involved in PE degradation and exploring the mechanisms by which environmental microorganisms directly participate in the PE degradation process is of great significance for better plastic pollution control and for addressing plastic pollution problems worldwide.

[0003] The biodegradation of recalcitrant synthetic polymers like PE microplastics by highly efficient microorganisms has garnered increasing attention. Microorganisms possess the ability to naturally transform or accumulate various compounds, including hydrocarbons (polycyclic aromatic hydrocarbons), pharmaceutical substances, and metals. Furthermore, the biodegradation of plastics by certain enzyme systems can lead to polymer breakdown into lower molecular weight, weaker mechanical properties, or further transformation into organic intermediates such as acids, alcohols, and ketones. These water-soluble pyrolysis products are absorbed by microbial cells, where they are metabolized. Aerobic metabolism produces carbon dioxide and water, while anaerobic metabolism produces carbon dioxide, water, and methane. Theoretically, these microorganisms can degrade PE plastics through their own metabolism. Therefore, researching and analyzing functional microorganisms for PE plastic degradation can effectively and thoroughly remove the environmental impact of plastic pollutants, ensuring environmental health and safety.

[0004] Aquabacterium is a rod-shaped, Gram-negative bacterium. It was first isolated from drinking water biofilms in Berlin in 1999. However, to date, no reports have been found regarding the ability of Aquabacterium strains to degrade PE. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings and deficiencies of the prior art and to provide a strain of Aquabacterium sp. A3, collected from Haigou Island, Guangzhou City, Guangdong Province, which was deposited at the Guangdong Provincial Center for Microbial Culture Collection on May 26, 2023, with accession number GDMCC No:63469.

[0006] Another object of the present invention is to provide the application of the above-mentioned Aquabacterium in the degradation of polyethylene plastics.

[0007] The objective of this invention is achieved through the following technical solution:

[0008] A strain of Aquabacterium, collected from Haigou Island in Guangzhou, Guangdong Province, was obtained through artificial enrichment culture, isolation and purification, and named Aquabacterium sp.A3.

[0009] The aforementioned *Aquabacterium* has the accession number GDMCC No:63469 and was deposited on May 26, 2023, at the Guangdong Provincial Center for Microbial Culture Collection, located at the Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province.

[0010] The aforementioned Aquabacterium cells are rod-shaped, Gram-negative, and have a cell size of 0.5–0.6 × 3.2–3.6 μm.

[0011] The nucleotide sequence of the 16S rRNA of the aforementioned Aquabacterium is shown in SEQ ID NO: 3.

[0012] The application of the aforementioned Aquabacterium in the degradation of polyethylene plastics.

[0013] The polyethylene plastic mentioned includes PE microplastic particles in water and the environment.

[0014] A method for screening highly efficient plastic-degrading bacteria includes the following steps:

[0015] Water samples were collected, and a certain amount of PE microplastics was added to the fresh water samples. The samples were then incubated for 30 days in a shaker at 150 rpm and 30°C. Microbial communities on the surface of the PE microplastics were separated by ultrasonication to obtain a microbial mixture. This mixture was evenly spread onto solid LB agar medium and incubated at 30°C in a biochemical incubator. After colonies grew, colonies of different morphologies were selected and isolated using the streak plate method. After repeated isolation and purification, single colonies were obtained. Highly efficient plastic-degrading bacteria were obtained through first-generation sequencing and validation experiments.

[0016] The present invention has the following advantages and effects compared with the prior art:

[0017] This invention provides an Aquabacterium strain, Aquabacterium sp. A3. Quantitative analysis using gas chromatography (GC) revealed that this strain Aquabacterium sp. A3 has the ability to mineralize PE, converting it into CO2. Fourier transform infrared spectroscopy (FTIR-ATR mode) analysis of the surface functional groups of the plastic showed that the addition of this strain Aquabacterium sp. A3 significantly altered the content and number of functional groups on the surface of PE microplastics, demonstrating that this bacterium has the ability to efficiently mineralize the surface of PE microplastics and has the potential to achieve biodegradation of microplastics in environments such as groundwater. Attached Figure Description

[0018] Figure 1 This is a scanning electron microscope image of Aquabacterium sp. A3 strain.

[0019] Figure 2 This is a phylogenetic analysis diagram of Aquabacterium sp. A3 strain.

[0020] Figure 3 This is a trend graph showing the mineralization of PE into CO2 by the Aquabacterium sp. A3 strain in Example 3.

[0021] Figure 4 This is the infrared spectrum of the microplastics before and after mineralization by the Aquabacterium sp. A3 strain in Example 3. Detailed Implementation

[0022] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0023] Unless otherwise specified in the following implementation plan, the test conditions are generally as per standard test conditions or the test conditions recommended by the reagent company. Unless otherwise specified, all materials and reagents used are commercially available.

[0024] Example 1: Isolation and purification of strains

[0025] (1) Water samples were collected from the water around Haigou Island in Guangzhou, Guangdong Province. 250 mL of water samples from Haigou Island were placed in a 500 mL conical flask, 20 g of PE microplastics (plastic particle size range between 3 and 1000 μm) were added, the flask was sealed with a sterile aerobic membrane, and the flask was placed in a 30℃ shaker at 150 rpm for incubation.

[0026] (2) After 30 days of culture, 1g of PE microplastics from the conical flask was transferred to a 5mL sterile centrifuge tube. The tube was washed three times with 0.9% sterile saline solution, then filled with 0.9% sterile saline solution. The centrifuge tube was sealed and sonicated for 15 minutes to obtain a microbial mixture on the surface of the PE microplastics. The microbial mixture was diluted at 1x, 10x, 100x, 1000x, and 10000x ratios, and then evenly spread onto solid LB medium. The medium consisted of 10g / L tryptone, 5g / L yeast, 10g / L NaCl, and 15g / L agar. After inoculation, the culture was placed in a biochemical incubator at 30℃. After bacterial growth, colonies of different morphologies were picked and isolated and purified using the streak plating method. These colonies were then streaked onto LB solid medium. This step was repeated four times to obtain single colonies, which were then stored at 4℃ for later use and preserved in ceramic bead culture tubes at -80℃.

[0027] Example 2: Identification of the strain

[0028] (1) Colony morphology

[0029] The strain in the single colony obtained in Example 1 was rod-shaped, Gram-negative, and the SEM image is shown below. Figure 1 As shown, the bacterial cell size is 0.5–0.6 × 3.2–3.6 μm.

[0030] (2) Molecular biological identification

[0031] DNA was extracted from the single colony obtained in Example 1 using a bacterial genomic DNA extraction kit. Then, the 16S rRNA gene sequence (SEQ.ID.NO.3) was amplified and sequenced using the universal primer pairs 27F (5'-agagtttgatcmtggctcag-3') and 1492R (5'-ggytaccttgttacgactt-3') (SEQ.ID.NO.2). The obtained DNA sequence was entered into GenBank and compared with all sequences in the database using the Blasten program.

[0032] A phylogenetic tree was constructed using the 16S rRNA gene sequence (e.g. Figure 2 As shown in the figure, the 16S rRNA of a single colony in Example 1 was found to have a similarity of 97.55% with that of Aquabacterium lacunae.

[0033] Based on the results of the above two aspects, it was determined that the strain isolated in Example 1 belonged to the genus Aquabacterium and was named Aquabacterium sp. A3.

[0034] Example 3: Mineralization properties of Aquabacterium sp. A3 on PE microplastics

[0035] The study on the mineralization properties of PE microplastics included three groups: a group with added bacteria, a group without added bacteria, and a sterilized group. The specific procedures were as follows:

[0036] PE pretreatment: All experimental plastics were washed three times with 75% alcohol, then sterilized overnight under ultraviolet light in a clean bench, and dried at room temperature for later use.

[0037] Inoculation group: 20 mg of UV-sterilized PE and 20 mL of MSM medium (composed of 1.5 g / L KH₂PO₄, 10.55 g / L Na₂HPO₄·12H₂O, 0.3 g / L NH₄Cl, and 0.1 g / L MgCl₂·6H₂O) were added to a 50 mL vial. The vial was autoclaved at 121 °C for 20 min. After cooling, 1 mL / L mixed vitamins and 1 mL / L trace elements were added. Aquabacterium sp. A3 was centrifuged from the liquid medium and inoculated into vials at 4000 rpm for 10 min. The vials were sealed with rubber stoppers and incubated at 30 °C with shaking at 150 rpm. The CO₂ concentration in the headspace was measured using gas chromatography at days 0, 1, 2, 3, 5, 7, and 10.

[0038] Sterilization group: 20 mg PE and 20 mL MSM medium were added to 50 mL vials (medium composition as above). Aquabacterium A3 was centrifuged from the liquid medium and inoculated into the vials. The vials were then autoclaved at 121°C for 20 min. After inoculation, the vials were sealed with rubber stoppers and incubated at 30°C with shaking at 150 rpm. The CO2 concentration in the system was measured using gas chromatography at days 0, 1, 2, 3, 5, 7, and 10.

[0039] Untreated group: Add 20 mg PE and 20 mL MSM medium (composition as above) to a 50 mL vial. Seal the vial with a rubber stopper and incubate at 30°C with shaking at 150 rpm. Detect the CO2 concentration in the system gas using gas chromatography at days 0, 1, 2, 3, 5, 7, and 10.

[0040] Plot the headspace CO2 concentration changes of the sterilized group, sterilized group, and no sterilized group as follows: Figure 4 As shown. Figure 3 In the middle: the X-axis represents time (days), with sampling times of 0 days, 1 day, 2 days, 3 days, 5 days, 7 days, and 10 days; the Y-axis represents the total CO2 concentration in the headspace of the microcosm system (ppm).

[0041] from Figure 3 It can be seen that in the unsterilized and sterilized groups, the CO2 concentration remained at around 500 ppm; while in the group with added bacteria, the CO2 concentration increased from around 600 ppm to around 2200 ppm, indicating that the PE mineralization reaction occurred from the introduction of the bacterial strain. Introducing the bacterial strain Aquabacterium sp. A3 into the PE-containing culture medium can significantly accelerate the PE mineralization reaction.

[0042] Infrared spectral characterization of PE microplastics before and after mineralization confirmed the oxidation phenomenon occurring on the plastic surface. Specific examples are as follows:

[0043] After separating the PE microplastics from water in the bacterial-added and unadded groups, the PE microplastics were placed in 10mL centrifuge tubes, filled with ultrapure water, sealed, and sonicated for 15 minutes to remove microorganisms adhering to the surface of the PE microplastics. The tubes were then rinsed with ultrapure water, and this process was repeated three times to obtain the PE microplastics in the bacterial-added and unadded groups. After freeze-drying, the microplastics were sent to a testing company, where changes in the functional groups on the surface of the PE microplastics were detected using Fourier Transform Infrared Spectroscopy (FTIR-ATR mode).

[0044] Figure 4 As can be seen, in the infrared results of the original PE plastic and the PE plastic with added bacteria, 2922 cm⁻¹ -1 and 2850cm -1 The peaks at 1466 cm⁻¹ represent the asymmetric and symmetric stretching vibrations of -CH₂⁻, respectively. -1 and 722cm -1 These are the bending vibrations of -CH2- and -(CH2), respectively. n - The in-plane bending vibration, which is a characteristic peak of polyethylene plastic. The difference is that on the surface of plastic with added pure bacteria, it is located at 3420 cm⁻¹. -1 The characteristic peak of the nearby -OH group is significantly enhanced, at 1146 cm⁻¹. -1 A relatively obvious characteristic peak of primary alcohol also appeared, at 1641 cm⁻¹. -1 The presence of functional groups, represented by carboxyl groups, and the appearance of these different characteristic peaks proves that the strain has a mineralizing effect on plastics, which leads to the destruction of the original structure of polyethylene plastic and the appearance of oxygen-containing functional groups on the surface of the plastic.

[0045] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A strain of *Aquabacterium*, characterized by: Name is Aquabacterium sp. A3, with accession number GDMCC No: 63469, was deposited on May 26, 2023, at the Guangdong Provincial Center for Microbial Culture Collection, located at the Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province.

2. The application of the Aquabacterium as described in claim 1 in the degradation of polyethylene plastics.

3. The application according to claim 2, characterized in that: The polyethylene plastic mentioned includes PE microplastic particles in water and the environment.

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