Application of escherichia coli in plastic degradation

Abstract

The application relates to the technical field of microbial application, and discloses application of escherichia coli in plastic degradation. The strain grows faster than normal bacteria in PP and PVC microplastic solutions, and can colonize and erode PP and PVC plastic films to survive by taking PP and PVC films as a nutrient source. The BL21 (DE3) escherichia coli provided by the application can effectively realize degradation of PP and PVC plastics, provides a strain resource for PP and PVC plastic biodegradation, and has a good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of microbial application technology, specifically involving a new application of BL21(DE3) Escherichia coli in plastic degradation. Background Technology

[0002] Polypropylene (PP) is the second largest source of waste plastics after polyethylene. Due to its structural stability, it does not decompose rapidly in the natural environment, and there are few reports on the biodegradation of PP or its use in supporting microbial growth. Polyurethane films can be degraded by *Pseudomonas aeruginosa* MTCC 7616, *Pseudomonas stutzeri*, *Bacillus subtilis*, and *Bacillus curvatureis* strains. *Bacillus curvatureis* strains showed the highest degradation rate, with a weight loss of approximately 2.5% after 12 months (Arkatkar et al., 2010). *Rhodococcus* strain 36 and *Bacillus* strain 27 showed weight losses of 6.4% and 4%, respectively, after 40 days of cultivation in polypropylene films (Auta et al., 2017). Pretreatment with oxidants or starch and mixing it into polypropylene, followed by exposure to *Bacillus non-neurobacterium* and *Brucea* strains, has proven to be an effective biodegradation strategy (Skariyachan et al., 2018). Jeon et al. (2021) demonstrated a novel polypropylene-degrading bacterium, Bacillus lysine, strain JJY0216, which resulted in a 4% weight loss of polypropylene after 26 days of exposure.

[0003] In terms of demand from the plastics industry, polyvinyl chloride (PVC) ranks third, after polyethylene and polypropylene, and is widely used in packaging. Regarding biodegradation, previous studies have detected plastic-degrading capabilities in Pseudomonas, Brevibacterium, and Micrococcus (Martins-Franchetti et al., 2010). Some potential PVC biodegrading bacteria are *Pseudomonas otitis media*, *Bacillus cereus*, and *Echinobacterium aviculare* (Anwar et al., 2016). Giacomucci et al. (Giacomucci et al., 2019) found that after culturing PVC with *Bacillus curvatureii* for 90 days, the average molecular weight of PVC decreased by 93.48% from 100%.

[0004] In summary, some bacteria may possess degradation capabilities for PP and PVC plastics. However, engineered bacteria commonly used in biology are modified from normal microorganisms, originating from nature and primarily used to assist scientific experiments, neglecting their inherent characteristics. Therefore, we propose that commonly used bacteria in biology may also affect recalcitrant plastics such as polypropylene (PP) and polyvinyl chloride (PVC). Verification revealed that *E. coli* BL21(DE3) can effectively degrade PP and PVC plastics, providing a new approach to the biodegradation of PP and PVC plastics through microbial degradation, with promising application prospects. Summary of the Invention

[0005] The purpose of this invention is to apply BL21(DE3) Escherichia coli to the degradation of plastics for the first time.

[0006] Furthermore, the plastics include PP and / or PVC.

[0007] Furthermore, the Escherichia coli mentioned includes BL21(DE3).

[0008] Furthermore, the Escherichia coli contains an antibiotic-resistant plasmid.

[0009] Furthermore, the Escherichia coli contains -Blunt E1 plasmid.

[0010] The specific application involves adding PP and / or PVC to the bacterial solution for degradation.

[0011] This invention departs from the traditional approach of searching for bacteria that can degrade plastic from plastic piles. Instead, it uses the concept that promotion is beneficial and beneficial is nutritious to determine whether existing bacteria have the ability to degrade plastic.

[0012] This invention also provides specific methods and procedures for finding potential plastic-degrading bacteria from known microorganisms, as well as new applications of Escherichia coli in plastic degradation.

[0013] This invention utilizes microplastic-stressed microorganisms to measure OD... 600 To reflect the growth of microorganisms, the growth status of microorganisms was determined by spot plate counting and scanning electron microscopy. Infrared spectroscopy was used to determine the response of microorganisms (extracellular polymers) to plastic particle stress. Microorganisms were placed in a culture medium containing plastic film and cultured for one week. The degradation of plastic by microorganisms was determined by scanning electron microscopy.

[0014] In summary, this invention determines whether plastic particles have a positive effect on BL21(DE3) Escherichia coli by detecting their impact, then examines the growth status of microorganisms with a positive effect, and finally uses plastic film to determine the degradation effect.

[0015] Compared with the prior art, the advantages and beneficial effects of this invention are as follows:

[0016] 1. This invention uses microplastic stress to qualitatively determine whether BL21(DE3) Escherichia coli has degradation potential, which is highly targeted.

[0017] 2. This invention is the first to apply engineered bacteria commonly used in biology to the degradation of PP and PVC plastics, which can serve as an effective research approach for solving current plastic pollution.

[0018] 3. This invention applies BL21(DE3) Escherichia coli to the degradation of PP and PVC plastics, and its degradation efficiency is good.

[0019] The present invention will now be described in further detail with reference to the accompanying drawings and specific embodiments. Attached Figure Description

[0020] Figure 1 The relative growth rate of BL21(DE3) Escherichia coli exposed to PP(a) and PVC(b) microplastics;

[0021] Figure 2 BL21(DE3) Escherichia coli survival rate was verified by spot plate counting method after exposure to PP and PVC microplastics;

[0022] Figure 3 : Validation of long-term stress on BL21(DE3) Escherichia coli exposed to PP and PVC microplastics;

[0023] Figure 4 The effects of microplastics on the growth of BL21(DE3) Escherichia coli were observed using scanning electron microscopy (SEM).

[0024] Figure 5 Fourier transform infrared (FTIR) analysis of extracellular polymeric changes in BL21(DE3) Escherichia coli;

[0025] Figure 6 Scanning electron microscope image and particle size distribution diagram of the PP microplastics used in this invention.

[0026] Figure 7 Scanning electron microscope image and particle size distribution map of the PVC microplastics used in this invention.

[0027] Figure 8 Scanning electron microscopy (SEM) of Escherichia coli BL21(DE3) on the degradation of plastic film. Detailed Implementation

[0028] The following experimental examples are further illustrative of the present invention and are not intended to limit the invention. Specific experimental conditions and methods are not specified in the following examples, and the techniques used are generally conventional methods well known to those skilled in the art.

[0029] Example 1: The unique response of Escherichia coli BL21(DE3) to microplastics was determined from three engineered bacteria of the genus *Agrobacterium*: *Agrobacterium* LBA4404, *Escherichia coli* DH5α, and *Escherichia coli* BL21(DE3). The specific steps are as follows:

[0030] Three engineered bacteria were inoculated into 50 mL of fresh LB liquid medium and cultured at 28°C until OD500. 600 =0.5, this is recorded as the initial concentration; PP and PVC microplastic particles are added to make the final concentration 1 mg / ml, and the OD of the bacterial solution is measured at time points of 0h, 0.5h, 1.0h, 1.5h, 2.0h, 2.5h, and 3.0h. 600 The value is used to qualitatively detect the relative growth rate by measuring the increase in each time period. Figure 1 ), the survival rate was calculated using the spot count method. Figure 2 ), and conduct long-term culture and observation of candidate bacteria ( Figure 3 The presence of PP and PVC promotes the growth of BL21(DE3) Escherichia coli, while having no significant inhibitory or promoting effect on the other two engineered bacteria. TLC also clearly showed that the total number of colonies was relatively higher in the presence of PP and PVC compared to the control. Long-term culture revealed that the bacterial solution containing PP and PVC grew significantly faster during both the growth and stationary phases. Higher bacterial concentrations indicate that PP and PVC microplastics have a potential promoting effect on the growth of BL21(DE3) Escherichia coli.

[0031] Example 2: The growth of E. coli after the addition of microplastics was detected. The specific steps are as follows:

[0032] After stressing *E. coli* with PP and PVC plastic particles for 24 h, the effects of microplastics on the morphology of *E. coli* were analyzed using scanning electron microscopy. First, the mixture was centrifuged at 1000×g for 10 min to remove most of the plastic material. Then, bacterial cells were collected by centrifugation at 12000×g for 10 min, washed twice with PBS buffer, and fixed with 2.5% glutaraldehyde solution for 3–4 h. The cells were then gradually dehydrated with a series of concentrations of ethanol (50%, 70%, 90%, 100%, and 100%) for 10 min each time. After dehydration, the bacteria were dried at the CO2 critical point and the bacterial cell morphology was observed using scanning electron microscopy (SEM). Figure 4 Fourier transform infrared spectroscopy was used to analyze changes in extracellular polymers in bacteria after exposure to plastic particles. Figure 5After adding PP and PVC plastics, there was no obvious damage to E. coli, and a large number of E. coli adhered to the microplastic particles. The plastic particles were coated with a series of complexes. Most of the PP was degraded into short rods and then attached to by E. coli. In the sample with added PVC, it was difficult to find obvious plastic particles, possibly because they were consumed by E. coli degradation or most of the plastic particles were removed by low-speed centrifugation. Fourier transform infrared spectroscopy detected E. coli with added plastic particles, and the lipid substances were very active, indicating that E. coli secreted a large amount of lipid substances to adhere to the microplastic particles.

[0033] Scanning electron microscope images and particle size distribution diagrams of the PP and PVC microplastics used in this invention are as follows: Figure 6 and 7 .

[0034] Example 3: Degradation verification of plastic film by BL21(DE3) Escherichia coli. The specific steps are as follows:

[0035] Plastic PP and PVC films were incubated for 7 days in culture media containing BL21(DE3) Escherichia coli and a control group without bacteria. The results were observed using a scanning electron microscope (SEM). Figure 8 The results showed that a large number of BL21(DE3) Escherichia coli colonized PP and PVC plastic films. After the ultrasonic cleaner completely removed the biomass attached to the plastic films, many severe cracks, fractures and deep pores were observed on the surface and even inside the PP and PVC films. SEM observation further confirmed that BL21(DE3) Escherichia coli has a significant ability to colonize and degrade PP and PVC, thus serving as a nutrient source for growth.

[0036] The above embodiments are only used to explain the inventive concept of the present invention, and are not intended to limit the protection of the present invention. Any non-substantial modifications and substitutions made to the relevant details of the present invention using this concept should fall within the protection scope of the present invention.

Claims

1. Application of Escherichia coli in plastic degradation; the plastic is PP and / or PVC; The Escherichia coli is BL21 (DE3); The Escherichia coli contains pEASY-Blunt E1 plasmid.

2. Use according to claim 1, characterized in that, PP and / or PVC are added into the bacterial solution for degradation.

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