CN114085799B - Spindle-shaped lysine bacillus preparation for degrading polystyrene plastic and its preparation - Google Patents

Abstract

The invention belongs to the technical field of biology, and relates to bacillus for biodegradation of polystyrene plastic, which is a fusiform lysine bacillus PS-02 strain. The invention also relates to a preparation of the bacillus fusiformis for degrading polystyrene plastic, which contains bacterial cells and/or spores of the bacillus fusiformis. The bacillus for biodegradable polystyrene plastic provided by the invention can be used for efficiently biodegrading polystyrene plastic films and microplastic, and has important application prospects in the aspect of biodegrading polystyrene plastic.

Description

Spindle-shaped lysine bacillus preparation for degrading polystyrene plastic and its preparation

technical field

The invention belongs to the technical field of microorganisms, and relates to a spindle-shaped lysine bacillus preparation for degrading polystyrene plastics and its preparation, in particular to a spindle-shaped lysine bacillus preparation for degrading polystyrene plastics and a preparation method thereof with application.

Background technique

The environmental problems caused by plastic products cannot be ignored, and the diffusion and migration of Microplastics (MPs) particles will form, causing various hazards in the global ecosystem. The main types of plastics that form microplastic particle pollutants are polystyrene (Polystyrene, PS), polyethylene (PE) and polypropylene (PP), among which microplastic particles formed by polystyrene have a particularly serious impact on the environment. It is the main microplastic pollutant in the middle and lower Yangtze River Basin and southern mangroves in my country, and accounts for 13% of the total microplastics in the freshwater environment.

Polystyrene microplastics have a certain degree of hindrance to the photosynthesis and growth of aquatic and terrestrial plants, and will accumulate as a persistent potential pollutant through the food web, affecting physiological and molecular reactions and hindering the immune response of organisms , leading to health risks such as infertility, obesity and cancer. Polystyrene microplastics are difficult to remove from the natural environment due to their small size and chemical inertness. At present, there are few studies on the decomposition and treatment of polystyrene microplastic pollutants. The use of biological methods to degrade plastics has the advantages of high efficiency, less pollution, and renewability, and provides a new idea for solving the problem of polystyrene microplastic pollution. Although it has been confirmed that PS can be degraded by microorganisms, there are few types of strains with relevant abilities that have been found so far. Eisaku et al. found that 5 strains of bacteria belong to Xanthomonas sp., Sphingomonas ( Sphingobacterium sp.) and Bacillus sp. STR-Y-O bacteria have a certain ability to degrade polystyrene strains, Rhodococcusruber isolated by Mor et al. High affinity, can form biofilms and may cause partial biodegradation of polystyrene plastics. Atiq et al. isolated from soil 6 strains including Pseudomonas aeruginosa attached to the surface of polystyrene plastic film. However, the above-mentioned bacteria are not satisfactory, or they can only partially degrade polystyrene plastics, or the degradation efficiency is not ideal, or they cannot degrade polystyrene microplastics at all.

Contents of the invention

One of the objectives of the present invention is to provide a spindle for biodegrading polystyrene plastics in order to solve the problems of incomplete degradation of polystyrene plastics, low degradation efficiency, and inability to degrade polystyrene microplastics in the prior art. Bacillus lysinica, which can rapidly and efficiently biodegrade polystyrene plastics in water environment.

The second object of the present invention is to provide a spindle-shaped lysine bacillus preparation for degrading polystyrene plastics. Composed of acid bacillus, it can efficiently bio-generate polystyrene plastic.

The third object of the present invention is to provide a method for cultivating the above-mentioned spindle-shaped lysine bacillus bacterial strain, by using this method to ferment and cultivate the above-mentioned bacterial strain, the culture obtained has higher optical density and cell colony forming unit ( CFU).

The fourth object of the present invention is to provide an application of the above-mentioned spindle-shaped lysine bacillus preparation in degrading polystyrene plastic films and microplastics in water environment.

To this end, the first aspect of the present invention provides a bacillus for biodegrading polystyrene plastics, said bacillus is Bacillus fusiformis lysine PS-02 strain, and its preservation number is CGMCC No.23975.

In some embodiments of the present invention, the bacterial cells and/or spores of the 0.01 g/mL Bacillus spindle-shaped lysinica PS-02 strain can reduce the water contact angle of polystyrene plastic film from From 97.0°±4.60° to 64.0°±3.62°, the infrared spectrum of polystyrene microplastics produces a new characteristic peak at the wavenumber of 800-1700 cm ^–1 , and scanning electron microscope detection shows that polystyrene microplastic particles are composed of The flat and smooth surface has changed to a form full of ravines and breakages.

The second aspect of the present invention provides a spindle-shaped lysine Bacillus preparation for degrading polystyrene plastics, which contains bacterial cells and/or spores of Bacillus as described in the first aspect of the present invention; preferably, the The preparation of Bacillus fusiformis contains the spores of Bacillus according to the first aspect of the present invention.

In some embodiments of the present invention, the spindle-shaped lysine bacillus preparation for degrading polystyrene plastic is a liquid preparation; preferably, in the liquid preparation for degrading polystyrene plastic, the bacillus The concentration of bacterial cells and/or spores is ≥1×10 ⁹ /mL, more preferably (1-3)×10 ⁹ /mL, even more preferably (2-3)×10 ⁹ /mL;

In some other embodiments of the present invention, the spindle-shaped Bacillus lysinus preparation for degrading polystyrene plastic is a solid powder preparation; preferably, in the solid powder preparation for degrading polystyrene plastic, the The content of bacterial cells and/or spores of Bacillus ≥ 1×10 ¹¹ /g, more preferably (1-3)×10 ¹¹ /g, still more preferably (2-3)×10 ¹¹ /g.

The third aspect of the present invention provides a method for preparing a spindle-shaped lysine bacillus preparation for degrading polystyrene plastics as described in the second aspect of the present invention, comprising:

Step B, inoculating the fermentation strain into the fermentation medium for fermentation culture to obtain a fermentation culture of Bacillus;

Step C, centrifuging the fermentation culture of Bacillus, harvesting the wet Bacillus cells containing bacterial cells and/or spores as a liquid preparation of Bacillus lysinica;

Wherein, the fermented strains are obtained from corresponding bacterial strains through seed cultivation;

The corresponding strains of the fermented species are the strains of Bacillus described in the first aspect of the present invention.

According to the present invention, the fermentation medium includes the following components in 1L of water in terms of 1L of water:

Glucose monohydrate 20-30g;

Malt powder 1-3g;

_{KH2PO4 1-3g} ;

MgSO ₄ •7H ₂ O 1-3g;

MgCl ₂ • 4H ₂ O 0.2-0.6g;

CaCl ₂ 0.5-1.5g;

Sodium chloride 5-10g;

Beef extract 15-25g;

Peptone 5-10g;

Yeast paste 5-10g;

Preferably, the pH value of the fermentation medium is 6.5-7.5; further preferably, in step B, the temperature of the fermentation culture is 30-35°C.

According to the present invention, the preparation method further includes step D after step C, after freeze-drying the wet bacillus cells containing bacterial cells and/or spores to obtain a solid powder preparation for degrading polystyrene plastics.

In the above step B, the optical density (OD680nm) of the fermentation culture is ≥ 45; the cell colony forming unit (CFU) of the fermentation culture is ≥ 2×10 ⁹ /mL.

The fourth aspect of the present invention provides a spindle-shaped lysine bacillus preparation for degrading polystyrene plastic as described in the second aspect of the present invention or the degraded polystyrene prepared by the preparation method as described in the third aspect of the present invention. Application of a preparation of Bacillus spindle-shaped lysinus for vinyl plastics in the degradation of polystyrene plastics.

According to the present invention, the application includes adding the spindle-shaped lysine bacillus preparation into the degradation medium containing polystyrene plastics, and degrading the polystyrene plastics.

Preferably, the polystyrene plastic includes environmental polystyrene plastic waste; further preferably, the polystyrene plastic includes environmental polystyrene plastic film waste, polystyrene microplastics.

In some embodiments of the present invention, the degradation medium is a liquid medium in which polystyrene plastic is the only carbon source, preferably, the degradation medium is calculated in 1L of water, and includes the following components in 1L of water :

NaHCO ₃ 0.2g;

(NH ₄ ) ₂ SO ₄ 1.0 g;

CaCO ₃ 0.1g;

Na ₂ HPO ₄ • 12H ₂ O 4.37g;

NaH ₂ PO ₄ • 2H ₂ O 1.22g;

FeSO ₄ •7H ₂ O 0.01g;

MgSO ₄ •7H ₂ O 0.01g;

CuSO ₄ • 5H ₂ O 0.001g;

MnSO ₄ • 5H ₂ O 0.001g;

ZnSO ₄ •7H ₂ O 0.001g;

Preferably, the pH value of the degradation medium is 7.0.

In other embodiments of the present invention, the dosage of the Bacillus fusiformis lysinus preparation is 0.01 g/mL, the degradation treatment temperature is 28-30° C., and the degradation treatment time is 4 weeks.

The present invention obtains polystyrene degrading bacterial strain-spindle-shaped lysinic acid bacillus PS-02 strain through separation and screening, can be effectively used in the biodegradation polystyrene, as polystyrene plastic film, granule, powder, adopts the present invention The strain degrades polystyrene plastic. After 30 days of treatment, there are obvious bio-erosion holes on the surface of the polystyrene membrane, the hydrophilicity of the membrane is significantly improved, and the surface is severely damaged. The degradation is green, low-cost, easy to operate, and suitable for natural treatment. Polystyrene waste, which is difficult to degrade in the environment, is of great significance to the protection of the ecological environment and human health.

Description of drawings

In order to make the present invention easy to understand, the present invention will be described below in conjunction with the accompanying drawings.

Fig. 1 shows the molecular phylogenetic tree of Lysinibacillus spindle-shaped PS-02 based on 16S rDNA.

Figure 2 shows the growth curve of Lysinibacillus fusiformis PS-02.

Fig. 3 shows the result of the particle size distribution of Bacillus lysinus spindle-shaped PS-02 used to degrade polystyrene microplastic samples in water.

Fig. 4 shows the results of infrared spectrum analysis of Bacillus lysinus spindle-shaped PS-02 used to degrade polystyrene microplastic samples in water.

Fig. 5 shows the scanning electron microscope analysis results of Bacillus lysinus spindle-shaped PS-02 used to degrade polystyrene microplastic samples in water.

Figure 6 shows the contact angle analysis results of Bacillus lysinus spindle-shaped PS-02 used to degrade polystyrene plastic film samples in water.

Fig. 7 shows the scanning electron microscope analysis results of Bacillus lysinus spindle-shaped PS-02 used to degrade polystyrene plastic film samples in water.

Culture preservation

The strain was isolated and identified by Beijing University of Science and Technology, and has been preserved in the General Microbiology Center of the China Committee for the Collection of Microbial Cultures (abbreviation: CGMCC; address: Institute of Microbiology, Chinese Academy of Sciences, No. 1, Beichen West Road, Chaoyang District, Beijing) and named after classification It is Lysinibacillus fusiformis, date of deposit: November 25, 2021, deposit number: CGMCC No.23975. In the present invention, the strain is named Lysinibacillus fusiformis strain PS-02 ( Lysinibacillus fusiformis strain PS-02).

Detailed ways

In order to make the present invention easy to understand, the present invention will be described in detail below. Before the invention is described in detail, however, it is to be understood that the invention is not limited to the particular embodiments described. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

Unless otherwise defined, all terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described.

Ⅰ. Terminology

The term "bacteria" in the present invention refers to living cells and/or dead cells of bacteria.

The term "spore" in the present invention refers to a dormant body formed by bacillus under certain conditions with strong stress resistance.

In the present invention, the term "Lysinibacillus fusiformis" and "Lysinibacillus fusiforme" or "Lysinibacillus fusiforme" or "Lysinibacillus fusiforme" can be used interchangeably.

The term "polystyrene microplastic" and "polystyrene nano-microplastic" in the present invention can be used interchangeably.

The "water" used in the culture medium or fermentation culture process, unless otherwise specified, refers to sterile pure water obtained by filtering through a 0.22 µ filter membrane.

Ⅱ. Implementation plan

As mentioned above, the existing bacteria that degrade polystyrene plastics are not satisfactory, and there are problems such as incomplete degradation of polystyrene plastics, low degradation efficiency, and inability to degrade polystyrene microplastics. In view of this, the inventors have conducted extensive research on the biodegradation of polystyrene plastics.

On the basis of long-term research on waste treatment, the inventor successfully domesticated and screened out a pure microbial strain that efficiently biodegrades polystyrene plastics from soil samples of environmental plastic waste. The enzymes produced by the bacteria can Catalytic degradation of polystyrene plastics, especially the efficient degradation of polystyrene microplastics, has important application prospects in the efficient biodegradation of polystyrene plastics.

Therefore, the first aspect of the present invention relates to the Bacillus for biodegrading polystyrene plastics.

The inventors first successfully screened out a strain of Bacillus from the soil samples of environmental plastic waste (that is, the soil in contact with the surface of plastic waste in domestic waste landfills), which can produce and catalyze the degradation of polystyrene plastics and polystyrene microplastics. enzyme. After extracting the genomic DNA, PCR amplification and molecular identification based on 16S rDNA sequencing, it was identified as the genus Lysinibacillus fusiformissp . Based on the above, the strain was identified and named as Lysinibacillus fusiformissp. Bacillus strain PS-02 ( Lysinibacillus fusiformis strain PS-02 ). The strain has been preserved in the General Microorganism Center of China Committee for Culture Collection of Microorganisms, and the preservation number is CGMCC No.23975.

The results of the study show that the bacterial cells and/or spores of the spindle-shaped lysinic bacillus PS-02 strain at a concentration of 0.01g/mL can change the water contact angle of the polystyrene plastic film from 97.0°±4.60° within 4 weeks. decreased to 64.0°±3.62° (see Figure 6); at the same time, Fourier transform infrared spectrometer characterization showed that the bacterial cells of the spindle-shaped lysinic bacillus PS-02 strain at a concentration of 0.01g/mL could make The infrared spectrum of polystyrene microplastics produces new characteristic peaks at wavenumbers of 800-1700 cm ^–1 (see Figure 4), and it can be concluded that the degradation of polystyrene microplastics produces C=O, CC, C=C, etc. Active groups, indicating that macromolecular polystyrene depolymerization or chain scission occurred, and new products were produced, and scanning electron microscopy showed that the surface of flat and smooth polystyrene microplastic particles appeared and covered with obvious grooves and damage ( See Figure 5), indicating that the polystyrene microplastics were significantly degraded.

Based on the above, the second to fourth aspects of the present invention further provide the use or application of the Bacillus for biodegrading polystyrene plastics described in the first aspect of the present invention.

Specifically, the second aspect of the present invention provides a spindle-shaped lysine bacillus preparation for biodegrading polystyrene plastics, which belongs to a microbial preparation for biodegrading polystyrene plastics, which contains The bacteria cells and/or spores of the bacillus.

In some preferred embodiments of the present invention, the preparation of Bacillus spindle-shaped lysinus contains spores of the Bacillus according to the first aspect of the present invention.

According to some embodiments of the present invention, the preparation of the biodegradable polystyrene plastic lysinylbacillus spindle-shaped is a liquid preparation.

In some embodiments of the present invention, in the liquid preparation for degrading polystyrene plastics, the concentration of the bacterial cells and/or spores of the Bacillus spores is ≥1×10 ⁹ /mL, more preferably (1-3 )×10 ⁹ /mL, more preferably (2-3)×10 ⁹ /mL.

According to other embodiments of the present invention, the biodegradable polystyrene plastic preparation of Bacillus fusiformis is a solid powder preparation.

In some embodiments of the present invention, the spindle-shaped lysine bacillus preparation for degrading polystyrene plastic is a solid powder preparation; preferably, in the solid powder preparation for degrading polystyrene plastic, the spore The content of bacterial cells and/or spores of Bacillus is ≥1×10 ¹¹ /g, more preferably (1-3)×10 ¹¹ /g, and still more preferably (2-3)×10 ¹¹ /g.

The third aspect of the present invention provides a method for preparing the spindle-shaped lysine bacillus preparation of biodegradable polystyrene plastic as described in the second aspect of the present invention, which includes:

Step B, inoculating the fermentation strain into the fermentation medium for fermentation culture to obtain a fermentation culture of Bacillus;

Step C, performing centrifugation on the fermentation culture of Bacillus, harvesting the wet Bacillus cells containing bacteria cells and/or spores as a spindle-shaped lysine bacillus preparation;

Wherein, the fermented strains are obtained from corresponding bacterial strains through seed cultivation.

As known to those skilled in the art, at present, 16S rRNA is often used internationally for molecular identification of bacteria, so 16S rRNA can be used for comparison of similarities to obtain their homology. A phylogenetic tree was constructed based on the 16S rDNA sequencing results. The results of sequence comparison and phylogenetic tree analysis showed that the degrading bacteria PS-02 was most similar to the spindle-shaped lysine bacillus JQ900544.1 (the sequence similarity was 99%), The strain is Lysinibacillus fusiformis . Fig. 1 shows the molecular phylogenetic tree based on 16S rDNA, and the bacillus of the present invention is the spindle-shaped lysine bacillus PS-02 strain. Therefore, the fermentation strains used in the present invention are not limited to the field isolates used in the present invention. 16S rDNA is the DNA sequence corresponding to the coding rRNA on the bacterial chromosome, and exists in the chromosomal genome of all bacteria.

Based on the above, it is easy to understand that in the present invention, the corresponding strain of the fermentation species is the Bacillus strain described in the first aspect of the present invention. That is, under the premise of not changing the 16S rDNA of the spindle-shaped lysinibacillus PS-02, those skilled in the art can obtain the spindle-shaped lysinylbacillus PS-02 of the present invention through simple screening or mutagenesis to obtain the Invent the 16S rDNA highly homologous strain of Bacillus spindle-shaped lysinus PS-02, and obtain corresponding strains with the same or similar functions of biodegrading polystyrene plastics.

In the above step C, the centrifugation treatment includes centrifuging the precipitate obtained from the liquid fermentation culture (that is, bacterial cells and/or spores of Bacillus), resuspending and washing with physiological saline, and then performing centrifugation Treat to obtain bacteria cells and/or spores of Bacillus.

The present invention has no particular limitation on the conditions of the centrifugation in the above step C. In some embodiments of the present invention, for example, the centrifugation of the to-be-separated product can be performed at 8000-10000 r/min for 10-20 minutes.

According to the method of the present invention, the fermentation culture is a shaker or fermentation tank fermentation culture of strains, and the fermentation strains are inoculated into the fermentation medium in the form of seed liquid. The inoculum amount of the seed liquid is 0.1%-1% (v/v); preferably the inoculum amount of the seed liquid is 0.2%-0.5% (v/v); further preferably the inoculum amount of the seed liquid is 0.2 %(v/v). In the seed solution, the colony forming unit (CFU) of the strain is (1-3)×10 ⁹ /mL.

Specifically, the above-mentioned fermentation medium includes the following components in 1L of water in terms of 1L of water:

Glucose monohydrate 20-30g;

Malt powder 1-3g;

_{KH2PO4 1-3g} ;

MgSO ₄ •7H ₂ O 1-3g;

MgCl ₂ • 4H ₂ O 0.2-0.6g;

CaCl ₂ 0.5-1.5g;

Sodium chloride 5-10g;

Beef extract 15-25g;

Peptone 5-10g;

Yeast paste 5-10g;

In order to obtain higher productivity of the spindle-shaped lysinic bacillus PS-02 strain wet thallus, the fermentation medium was optimized, and the results showed that the preparation of the fermentation medium according to the following composition was beneficial to the fermentation culture, and the fermentation culture Based on 1L of water, including the following components in 1L of water:

Glucose monohydrate 20g;

Malt powder 1g;

KH ₂ PO ₄ 1g;

MgSO ₄ •7H ₂ O 1g;

MgCl ₂ • 4H ₂ O 0.2g;

CaCl ₂ 0.5g;

Sodium chloride 5g;

15g beef extract;

Peptone 5g;

Yeast paste 5g;

In some embodiments of the present invention, 40% (wt/v) sodium hydroxide solution and 36% (v/v) hydrochloric acid solution are used to adjust the initial pH value of the fermentation medium, and the pH value of the fermentation medium is 6.5-7.5, preferably 6.8-7.2, more preferably 7.2.

According to some embodiments of the present invention, the preparation method of the bacillus involved in the present invention also includes the step A of seed cultivation before step B: picking the single clone of the spindle-shaped lysine bacillus PS-02 strain provided by the present invention The colonies were inoculated into 100 mL of fermentation liquid medium, and cultured on a shaking table at a temperature of 35 °C and a rotation speed of 200 r/min for 2 days to obtain fermentation strains (seed liquid).

The inventors studied the effects of different temperatures on the growth of Lysinibacillus spindle-shaped PS-02, and found that at a temperature of 30-35°C, preferably at 35°C, Lysinibacillus spindle-shaped PS-02 grew faster.

According to the present invention, the preparation method further includes step D after step C, after freeze-drying the wet bacillus cells containing bacterial cells and/or spores to obtain a solid powder preparation for degrading polystyrene plastics.

In the above step B, the optical density (OD680nm) of the fermentation culture is ≥ 45; the cell colony forming unit (CFU) of the fermentation culture is ≥ 2×10 ⁹ /mL.

The fourth aspect of the present invention provides a spindle-shaped lysine bacillus preparation of biodegradable polystyrene plastic according to the second aspect of the present invention or the biodegradable polystyrene prepared by the preparation method described in the third aspect of the present invention. The application of the spindle-shaped lysine bacillus preparation of vinyl plastic in degrading polystyrene plastics can be understood as utilizing the spindle-shaped lysine bacillus preparation of biodegradable polystyrene plastic described in the second aspect of the present invention or According to the preparation method described in the third aspect of the present invention, the method for degrading polystyrene plastics by the preparation of Bacillus spindle-shaped lysine for biodegrading polystyrene plastics.

In the present invention, the polystyrene plastic includes environmental polystyrene plastic waste; further preferably, the polystyrene plastic includes environmental polystyrene plastic film waste and polystyrene microplastic.

According to the present invention, the application includes adding the spindle-shaped lysine bacillus preparation into the degradation medium containing polystyrene plastics, and degrading the polystyrene plastics.

In some specific embodiments of the present invention, the degradation medium is a liquid medium in which polystyrene plastic is the only carbon source, preferably, the degradation medium is calculated in 1L of water, and includes the following in 1L of water Components:

NaHCO ₃ 0.2g;

(NH ₄ ) ₂ SO ₄ 1.0 g;

CaCO ₃ 0.1g;

Na ₂ HPO ₄ • 12H ₂ O 4.37g;

NaH ₂ PO ₄ • 2H ₂ O 1.22g;

FeSO ₄ •7H ₂ O 0.01g;

MgSO ₄ •7H ₂ O 0.01g;

CuSO ₄ • 5H ₂ O 0.001g;

MnSO ₄ • 5H ₂ O 0.001g;

ZnSO ₄ •7H ₂ O 0.001g;

Preferably, the pH value of the degradation medium is 7.0.

Further preferably, the dosage of the Bacillus fusiformis lysinus preparation is 0.01 g/mL, the degradation treatment temperature is 28-30° C., and the degradation treatment time is ≥4 weeks.

III. Relevant detection methods in the present invention

(1) In the present invention, the concentration of cells and/or spores is determined by the following method:

For the determination of the concentration of Lysinobacillus spindle-shaped PS-02 cells and/or spores, take the culture of Bacillus spindle-shaped Lysinica PS-02, dilute a certain number of times with physiological saline, and directly use flow cytometry (Germany SYSMEX) to determine the concentration of cells and/or spores therein.

(2) In the present invention, the crude enzyme protein concentration is determined by the following method:

Take the cell-free extract of Bacillus spindle-shaped lysinus PS-02, dilute it by a certain number of times with phosphate buffer solution, add Coomassie Brilliant Blue G-250 dye reagent in proportion to react for 10 minutes, and use a 722S visible spectrophotometer (Shanghai Prism light) at 595nm to measure the absorbance, using the standard curve method to calculate the protein concentration.

Ⅲ. Example

The present invention will be described in detail below through specific examples. The experimental methods described below are routine laboratory methods unless otherwise specified. The experimental materials described below can be obtained from commercial sources unless otherwise specified.

(1) In the following examples, the composition of the fermentation broth of Bacillus fusiformis PS-02 is (per liter): 20g of glucose monohydrate, 1g of malt extract powder, 1g of KH ₂ PO ₄ , MgSO ₄ • 7H ₂ O 1g, MgCl ₂ • 4H ₂ O 0.2g, CaCl ₂ 0.5g, sodium chloride 5g, beef extract 15g, peptone 5g, yeast extract 5g, using 40% (wt/v) sodium hydroxide solution and 36 % (v/v) hydrochloric acid solution to adjust the initial pH of the fermentation medium to 7.2. Add 100 ml of the prepared growth medium into a 500 ml triangular flask, sterilize at high temperature and high pressure (121°C) for 20 minutes, and then sterilize for another 20 minutes under ultraviolet irradiation in a clean bench.

(2) In the following examples, the composition of the inorganic salt liquid medium (pH=7.0) used for degradation treatment is (per liter):

NaHCO ₃ 0.2g;

(NH ₄ ) ₂ SO ₄ 1.0 g;

CaCO ₃ 0.1g;

Na ₂ HPO ₄ • 12H ₂ O 4.37g;

NaH ₂ PO ₄ • 2H ₂ O 1.22g;

FeSO ₄ •7H ₂ O 0.01g;

MgSO ₄ •7H ₂ O 0.01g;

CuSO ₄ • 5H ₂ O 0.001g;

MnSO ₄ • 5H ₂ O 0.001g;

ZnSO ₄ •7H ₂ O 0.001 g.

Embodiment 1: Preparation of spindle-shaped lysinyl bacillus liquid preparation

(1) Preparation of seed solution: Pick a single clone of Lysinobacillus fusiformis PS-02 and inoculate it into 100ml of fermentation liquid medium, culture it on a shaking table at a temperature of 35°C and a rotation speed of 200r/min for 2 days to obtain the fermentation bacteria species (seed solution), in the seed solution, the colony forming unit (CFU) of the strain is 3×10 ⁹ /mL.

(2) Spindle-shaped lysine Bacillus fermentation culture:

Under sterile conditions in a clean bench, inoculate 0.2% (v/v) Bacillus spindle-shaped lysine PS-02 bacterial solution in the liquid medium of the Erlenmeyer flask, and the fermentation culture is a shaker fermentation culture in which the strain is free. Fermentation strains are inoculated into the fermentation medium in the form of seed liquid, and the fermentation culture is obtained after batch cultivation at a temperature of 35°C and a shaker speed of 200 rpm for 2 days;

Detect the optical density (OD _680nm ) of the culture every 6 hours during the fermentation process, measure 3 times each time and take the average value, and draw the growth curve of the degrading bacteria (see Figure 2). After 48 hours of fermentation, the optical density of the fermentation culture (OD _680nm ) ≥ 50; cell colony forming units (CFU) of the culture ≥ 3×10 ⁹ /mL. The wet thalline obtained by separating the fermented culture is prepared to obtain the bacterium agent.

(3) Cell separation:

After centrifugation (8000 r/min, 10 minutes), the supernatant was discarded to harvest the cells and/or spores of Bacillus spindle-shaped Lysinica PS-02 as a liquid preparation of Lysinica spindle-shaped.

Example 2: Degradation application of polystyrene microplastics in water

(1) Degradation treatment

Add 50mL inorganic salt liquid medium (without carbon source) and polystyrene microplastics (1.0g) with a particle size of 80nm or 100μm after ultraviolet sterilization to a 200mL Erlenmeyer flask to form a composition with polystyrene plastic as the only carbon source Inorganic salt liquid medium. Add 0.5 g of the plastic-degrading bacterial agent produced by fermentation into the Erlenmeyer flask, place it in a shaker at 35° C. and 200 r/min for cultivation, and test the degradation performance after 4 weeks.

(2) Sample treatment: Stir and wash the collected liquid medium with 2.0% sodium dodecylsulfonate (SDS) aqueous solution for 2 hours to remove the colony attached to the microplastic, wash it with water, and place it in a 65°C refrigerator. Dry in oven.

(3) Measure the particle size of the sample:

Particle size analyzer: The working principle of laser particle size analyzer is dynamic light scattering (Dynamic light scattering, DLS), which is a physical characterization method for measuring particle size distribution in solution or suspension, and can also be used to measure concentrated aggregates Behavior of complex fluids such as solid solutions. By illuminating the particle with a laser, the angle-specific scattering intensity will fluctuate over time, which can be detected using a sensitive avalanche photodiode detector (APD). Detection was performed on a Zetasizer Nano ZS90 laser particle size analyzer with a measurement range of 20 nm to 900 nm using dynamic light scattering (DLS). The intensity variation is analyzed with a digital autocorrelator, which generates a correlation function. The curves can be analyzed to give size and size distribution, see Figure 3.

From the detection results of the laser particle size analyzer, it can be known that microplastics have undergone significant agglomeration under the action of degrading bacteria, from the initial 80 nanometers to 350 nanometers. Considering that the particle size of bacteria is about micron level , so the 350-nanometer particle is not a bacterial cell, but a microplastic particle that is agglomerated under the action of enzymes and proteins secreted by bacteria. From this, it can be inferred that the degrading bacteria can secrete related enzymes or proteins. The smaller microplastics are aggregated and adhered together to reduce the dispersion of microplastics in the water environment, so as to achieve the effect of adsorption, enrichment and degradation, and can be applied to the biological purification carrier of water bodies.

(4) Fourier transform infrared spectroscopy (FTIR) analysis:

Samples were measured using Fourier transform infrared spectroscopy (FTIR) on a Nicolet iS50 FTIR spectrometer. The spectral recording range is 400–4000cm ^–1 , scanned at least 16 times, and the spectral resolution is 0.48cm ^–1 . Peaks were identified using OMNIC software. And according to the corresponding peak number and peak area to determine the content of metabolites, the results are shown in Figure 4.

According to the characterization results of Fourier transform infrared spectroscopy, it can be known that the functional groups of the microplastics after the treatment of the degrading bacteria have changed significantly. Compared with the control group, the products after the treatment of the bacteria have a wave number of ^800-1700 cm New characteristic peaks are generated, and it can be concluded that plastic degradation produces active groups such as C=O, CC, and C=C. Therefore, it can be concluded that polystyrene has undergone obvious degradation under the treatment of bacterial agents, macromolecular polymers have undergone depolymerization or chain scission, and new products have been produced.

(5) Scanning Electron Microscope (SEM):

Select a small amount of samples for brittle fracture with liquid nitrogen, fix them on the metal sample stage with double-sided adhesive tape, put the fixed samples in a vacuum evaporator to vacuum and spray gold for 25s to improve the conductivity and secondary electron yield. Place the sample in a scanning electron microscope, observe with an accelerating voltage of 2KV, select different magnifications, and shoot at a 360° angle to take representative microstructure photos, as shown in Figure 5.

From the detection results of the scanning electron microscope, it can be known that after the polystyrene microplastic particles were treated with the bacterial agent of polystyrene plastic-degrading bacteria, obvious grooves and damage appeared on the surface of the microplastic particles, while the surface of the microplastic particles in the control group smoother. It shows that during the growth process of the bacterial agent, polystyrene can be used as the only carbon source to grow, and by eroding the plastic particles, destroying its surface structure, and then having a significant degradation effect on the microplastic particles.

Embodiment 3: Degradation application of polystyrene plastic film in water body

(1) Degradation treatment

Degradation application: Add 50mL inorganic salt liquid culture medium (no carbon source) and several polystyrene film sheets (1.0g) after ultraviolet sterilization to a 200mL Erlenmeyer flask to form a polystyrene plastic as the only carbon source Inorganic salt liquid medium. Add 0.5 g of the plastic-degrading bacterial agent produced by fermentation into the Erlenmeyer flask, place it in a shaker at 35° C. and 200 r/min for cultivation, and test the degradation performance after 4 weeks.

(2) Determination of hydrophobic contact angle:

Stir and wash the collected polystyrene plastic film with 2.0% sodium dodecylsulfonate (SDS) aqueous solution for 2 hours, wash off the colony attached to the plastic film, wash it with water, and place it in an oven at 65°C to dry. The OCA20 optical contact angle tester measures the contact angle of water droplets on the surface of microplastics. During the measurement, drop the particle liquid on a glass slide, heat and dry it for measurement, the drop volume of the micro-syringe is 5 μL, measure 5 different points for each sample, and take the average value. The results are shown in Figure 6.

From the results of the water contact angle experiment, it can be seen that after the polystyrene plastic film is treated with the bacterial agent of polystyrene plastic-degrading bacteria, the water contact angle on the film surface decreases from 97.0°±4.60° to 64.0°±3.62°, and the hydrophobicity of the plastic surface significantly decreased, and the hydrophilicity was improved, further illustrating that the degrading bacteria have high-efficiency degradation ability on polystyrene plastics, can reduce the hydrophobicity of plastics, and accelerate the biodegradation of plastics.

(3) Elemental analysis:

Stir and wash the collected polystyrene plastic film with 2.0% sodium dodecylsulfonate (SDS) aqueous solution for 2 hours, wash off the colony attached to the plastic film, wash it with water, and place it in an oven at 65°C to dry. Use Elemental UNICUBE Elemental Analyzer, CHNS mode: the sample is burned and converted into CO ₂ , H ₂ O, N ₂ and SO ₂ in pure oxygen, and then separated by a chromatographic column for thermal conductivity detection to measure the C in the sample. , H, N, S content, the results are as follows:

Group N(%) C(%) H(%);

Theoretical value 0 92.3 7.69;

Control group 0 91.9 7.37;

Experimental group 0 86.5 7.55;

It can be shown from the elemental analysis test results that after the polystyrene plastic film is treated with the microbial agent of polystyrene plastic-degrading bacteria, compared with the control group, the content of C element in the treated plastic is significantly reduced by 5.4%, indicating that the bacterial agent During the growth process, polystyrene can be used as the only carbon source for growth, and the reduced C element may be decomposed into CO ₂ under the metabolism of degrading bacteria. The results show that degrading bacteria can significantly degrade polystyrene plastic films. .

(4) Scanning Electron Microscope (SEM):

Stir and wash the collected polystyrene plastic membrane with 2.0% sodium dodecylsulfonate (SDS) aqueous solution for 0.5h, wash off the culture medium attached to the plastic membrane, wash it with water, and place it in a 2.5% Stored in glutaraldehyde solution. Select a small amount of samples for brittle fracture with liquid nitrogen, fix them on the metal sample stage with double-sided adhesive tape, put the fixed samples in a vacuum evaporator to vacuum and spray gold for 25s to improve the conductivity and secondary electron yield. Place the sample in a scanning electron microscope, observe with an accelerating voltage of 2KV, select different magnifications, and shoot at a 360° angle to take representative microstructure photos, as shown in Figure 7.

From the detection results of the scanning electron microscope, it can be known that after the polystyrene plastic film is treated with the bacterial agent of the polystyrene plastic degrading bacteria, the degrading bacteria have an obvious biotransformation effect on the plastic film, and the surface of the plastic film is obviously broken in many places. And there are a lot of bacterial cells attached to the broken part, and the bacterial cells grow well, which shows that the bacterial agent can grow with polystyrene as the only carbon source during the growth process. By eroding the plastic particles, destroying its surface structure, and further degrading the polystyrene plastic The film undergoes significant degradation.

It should be noted that the above-mentioned embodiments are only used to explain the present invention, and do not constitute any limitation to the present invention. The invention has been described with reference to typical embodiments, but the words which have been used therein are words of description and explanation rather than words of limitation. The present invention can be modified as prescribed within the scope of the claims of the present invention, and the present invention can be revised without departing from the scope and spirit of the present invention. Although the invention described therein refers to specific methods, materials and examples, it is not intended that the invention be limited to the specific examples disclosed therein, but rather, the invention extends to all other methods and applications having the same function.

Claims (15)

1. A bacillus for biologically degrading polystyrene plastic is a fusiform lysine bacillusLysinibacillus fusiformis) PS-02 strain with preservation number of CGMCC No.23975.

2. A preparation of a polystyrene plastic-degrading B.fusiformis comprising the bacterial cell and/or spore of the Bacillus of claim 1.

3. The bacillus fusiformis formulation of claim 2, wherein the bacillus fusiformis formulation contains spores of the bacillus of claim 1.

4. The preparation of B.fusiformis according to claim 2 or 3, wherein,

the preparation of the fusiform lysine bacillus for degrading polystyrene plastic is a liquid preparation; in the liquid preparation of the degradable polystyrene plastic, the concentration of bacterial cells and/or spores of the bacillus is more than or equal to 1 multiplied by 10 ⁹ /mL；

Alternatively, the preparation of the fusiform lysine bacillus for degrading polystyrene plastic is a solid powder preparation; in the solid powder preparation of the degradable polystyrene plastic, the content of bacterial cells and/or spores of the bacillus is more than or equal to 1 multiplied by 10 ¹¹ /g。

5. The preparation of B.fusiformis according to claim 4, wherein,

in the liquid preparation of the degradable polystyrene plastic, the concentration of bacterial cells and/or spores of the bacillus is (1-3) multiplied by 10 ⁹ /mL；

Alternatively, in the solid powder preparation of the degraded polystyrene plastic, the content of the bacterial cells and/or spores of the bacillus is selected to be (1-3) ×10 ¹¹ /g。

6. The preparation of B.fusiformis according to claim 5, wherein,

in the liquid preparation of the degradable polystyrene plastic, the concentration of bacterial cells and/or spores of the bacillus is (2-3) multiplied by 10 ⁹ /mL；

Alternatively, in the solid powder formulation of the degraded polystyrene plastic, the bacillusThe content of bacterial cells and/or spores is selected from (2-3) ×10 ¹¹ /g。

7. A method of preparing the polystyrene plastic degrading bacillus fusiformis formulation of any one of claims 2-6, comprising:

step B, inoculating a fermentation strain into a fermentation medium for fermentation culture to obtain a fermentation culture of bacillus;

step C, carrying out centrifugal separation treatment on a fermentation culture of bacillus to obtain bacillus wet thalli containing bacterial cells and/or spores;

wherein the fermentation strain is obtained by seed culture of corresponding strain; the corresponding strain of the fermentation strain is the strain of bacillus according to claim 1.

8. The method of claim 7, wherein the fermentation medium comprises the following components in 1L of water, based on 1L of water:

20-30g of dextrose monohydrate;

1-3g of malt extract powder;

KH ₂ PO ₄ 1-3g；

MgSO ₄ •7H ₂ O 1-3g；

MgCl ₂ •4H ₂ O 0.2-0.6g；

CaCl ₂ 0.5-1.5g；

5-10g of sodium chloride;

15-25g of beef extract;

5-10g of peptone;

5-10g of yeast extract;

the pH value of the fermentation medium is 6.5-7.5.

9. The method according to claim 8, wherein in the step B, the fermentation culture is performed at a temperature of 30 to 35 ℃.

10. The preparation method according to any one of claims 7 to 9, further comprising a step D after the step C, of freeze-drying bacillus wet cells containing bacterial cells and/or spores to obtain a solid powder preparation of degraded polystyrene plastic.

11. Use of a preparation of a lysine bacillus fusiformis degrading polystyrene plastics according to any one of claims 2 to 6 for degrading polystyrene plastics; wherein, the application comprises adding the preparation of the lysine bacillus fusiformis into a degradation culture medium containing polystyrene plastic, and carrying out degradation treatment on the polystyrene plastic; the polystyrene plastic comprises environmental polystyrene plastic waste.

12. The use according to claim 11, wherein the polystyrene plastic comprises environmental polystyrene plastic film waste and polystyrene microplastic.

13. The use according to claim 12, wherein the degradation medium is a liquid medium with polystyrene plastic as sole carbon source.

14. The use according to claim 13, wherein the degradation medium comprises the following components in 1L of water, based on 1L of water:

NaHCO ₃ 0.2g；

(NH ₄ ) ₂ SO ₄ 1.0g；

CaCO ₃ 0.1g；

Na ₂ HPO ₄ •12H ₂ O 4.37g；

NaH ₂ PO ₄ •2H ₂ O 1.22g；

FeSO ₄ •7H ₂ O 0.01g；

MgSO ₄ •7H ₂ O 0.01g；

CuSO ₄ •5H ₂ O 0.001g；

MnSO ₄ •5H ₂ O 0.001g；

ZnSO ₄ •7H ₂ O 0.001g；

the pH value of the degradation culture medium is 7.0.

15. The use according to any one of claims 11 to 14, wherein the amount of the preparation of bacillus fusiformis is 0.01g/mL, the degradation treatment temperature is 28-30 ℃, and the degradation treatment time is not less than 4 weeks.

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