CN114085799A - Spindle-shaped lysine bacillus preparation for degrading polystyrene plastic and its preparation - Google Patents
Spindle-shaped lysine bacillus preparation for degrading polystyrene plastic and its preparation Download PDFInfo
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Abstract
本发明属于生物技术领域，涉及一种用于生物降解聚苯乙烯塑料的芽孢杆菌，所述芽孢杆菌为纺锤形赖氨酸芽孢杆菌PS‑02株。本发明还涉及一种降解聚苯乙烯塑料的纺锤形赖氨酸芽孢杆菌制剂，该纺锤形赖氨酸芽孢杆菌制剂含有上述芽孢杆菌的菌细胞和/或芽孢。本发明所提供的用于生物降解聚苯乙烯塑料的芽孢杆菌能够高效生物降解聚苯乙烯塑料薄膜及微塑料，在生物降解聚苯乙烯塑料方面具有重要的应用前景。
The invention belongs to the field of biotechnology, and relates to a bacillus for biodegrading polystyrene plastics, wherein the bacillus is a spindle-shaped lysine bacillus PS-02 strain. The present invention also relates to a fusiform lysine bacillus preparation for degrading polystyrene plastic, the fusiform lysine bacillus preparation containing the bacterial cells and/or spores of the above-mentioned bacillus. The bacillus for biodegrading polystyrene plastics provided by the invention can efficiently biodegrade polystyrene plastic films and microplastics, and has important application prospects in biodegrading polystyrene plastics.
Description
Technical Field
The invention belongs to the technical field of microorganisms, relates to a lysine bacillus fusiformis preparation for degrading polystyrene plastics and a preparation method thereof, and particularly relates to a lysine bacillus fusiformis preparation for degrading polystyrene plastics and a preparation method and application thereof.
Background
Environmental problems associated with plastic products have been neglected, and Micro Plastic (MPs) particles are formed to diffuse and migrate, causing various hazards in the global ecosystem. The main plastic types forming the micro plastic particle pollutants are Polystyrene (PS), Polyethylene (PE) and polypropylene (PP), wherein the micro plastic particles formed by the Polystyrene have serious influence on the environment, and the micro plastic pollutants are main micro plastic pollutants in the lower watershed of the Yangtze river in China, the mangrove forest in the south and the like and account for 13 percent of the total amount of the micro plastic in the fresh water environment.
The polystyrene micro plastic has certain effect of inhibiting photosynthesis and growth of aquatic and terrestrial plants, and can be used as a persistent potential pollutant to accumulate through a food net to influence physiological and molecular reactions so as to inhibit the immune response of organisms, thereby causing health risks such as sterility, obesity, cancer and the like. Polystyrene microplastics are difficult to remove from the natural environment due to their small size and chemical inertness. Currently, there is less research on the decomposition treatment of polystyrene microplastic pollutants. The plastic degraded by the biological method has the advantages of high efficiency, small pollution, reproducibility and the like, and provides a new idea for solving the problem of pollution of the polystyrene micro-plastic. Although it has been demonstrated that PS can be degraded by microorganisms, a relatively small number of strains have been found to date, Eisaku et al found that 5 strains of bacteria belonging to the genera Xanthomonas (Xanthomonas sp.), Sphingomonas (Sphingobacterium sp.) and Bacillus (Bacillus sp. STR-Y-O) have a certain ability to degrade polystyrene-competent strains, and Mor et al isolated Rhodococcus (Rhodococcus ruber) of the genus Actinomyces has a high affinity for polystyrene, can form biofilms, and may lead to partial biodegradation of polystyrene plastics. Atiq et al isolated 6 strains of Pseudomonas aeruginosa (Pseudomonas aeruginosa) adhered to the surface of a polystyrene plastic film from the soil. However, none of these bacteria is satisfactory, or only partially degrades polystyrene plastics, or the degradation efficiency is not ideal, or the polystyrene microplastics cannot be degraded at all.
Disclosure of Invention
One of the purposes of the invention is to provide lysine bacillus fusiformis for biodegradation of polystyrene plastic aiming at the problems of incomplete degradation of polystyrene plastic, low degradation efficiency, incapability of degrading polystyrene micro plastic and the like in the prior art, and the strain can quickly and efficiently biodegrade polystyrene plastic in a water environment.
The other purpose of the invention is to provide a lysine bacillus fusiformis preparation for degrading polystyrene plastics, which is composed of the lysine bacillus fusiformis for biologically degrading polystyrene plastics and can efficiently degrade polystyrene plastics.
It is another object of the present invention to provide a method for culturing the above-mentioned lysenin fusiformis strain, by which the above-mentioned strain is fermentatively cultured to obtain a culture having a high optical density and a high number of cell Colony Forming Units (CFU).
The fourth purpose of the invention is to provide the application of the lysine bacillus fusiformis preparation in degrading polystyrene plastic films and micro-plastics in a water environment.
Therefore, the invention provides a bacillus for biologically degrading polystyrene plastics, wherein the bacillus is a fusiform lysine bacillus PS-02 strain, and the preservation number of the bacillus is CGMCC No. 23975.
In some embodiments of the invention, the concentration of 0.01g/mL of the bacterial cells and/or spores of the lysine bacillus fusiformis PS-02 strain can reduce the water contact angle of the polystyrene plastic film from 97.0 +/-4.60 degrees to 64.0 +/-3.62 degrees within 4 weeks, so that the infrared spectrum of the polystyrene micro plastic is 800-1700 cm in wave number–1New characteristic peaks are generated, and scanning electron microscope detection shows that the polystyrene micro plastic particles are changed into a form of filling gullies and damages from flat and smooth surfaces.
In a second aspect, the present invention provides a lysine bacillus fusiformis preparation for degrading polystyrene plastic, which contains bacterial cells and/or spores of the bacillus according to the first aspect of the present invention; preferably, the lysine bacillus fusiformis preparation contains spores of the bacillus according to the first aspect of the invention.
In some embodiments of the invention, the polystyrene plastic-degrading lysinibacillus fusiformis formulation is a liquid formulation; preferably, in the liquid preparation for degrading polystyrene plastics, the concentration of the bacterial cells and/or spores of the bacillus is more than or equal to 1 x 109PermL, more preferably (1-3). times.109PermL, more preferably (2-3). times.109/mL；
In other embodiments of the present invention, the polystyrene plastic-degrading lysinibacillus fusiformis formulation is a solid powder formulation; preferably, in the solid powder preparation for degrading polystyrene plastics, the content of bacterial cells and/or spores of the bacillus is more than or equal to 1 x 1011(1-3). times.10 is more preferable11(2-3). times.10 is more preferable11/g。
In a third aspect of the present invention, there is provided a method for preparing a lysine bacillus fusiformis preparation for degrading polystyrene plastic according to the second aspect of the present invention, comprising:
b, inoculating the fermentation strain into a fermentation culture medium for fermentation culture to obtain a fermentation culture of bacillus;
step C, carrying out centrifugal separation treatment on the fermentation culture of the bacillus, and harvesting bacillus wet thalli containing germ cells and/or spores to be used as a hammer-shaped lysine bacillus liquid preparation;
wherein the fermentation strain is obtained by seed culture of corresponding strains;
the corresponding strain of the fermenting species is a strain of bacillus according to the first aspect of the invention.
According to the invention, the fermentation medium comprises, based on 1L of water, the following components in 1L of water:
20-30g of monohydrate glucose;
1-3g of malt extract powder;
KH2PO4 1-3g；
MgSO4•7H2O 1-3g；
MgCl2•4H2O 0.2-0.6g；
CaCl2 0.5-1.5g；
5-10g of sodium chloride;
15-25g of beef extract;
5-10g of peptone;
5-10g of yeast extract;
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 ℃.
According to the invention, the preparation method also comprises a step D after the step C, and the solid powder preparation for degrading the polystyrene plastic is obtained after the bacillus wet thalli containing the bacterial cells and/or the spores are freeze-dried.
In the step B, the optical density (OD 680 nm) of the fermentation culture is not less than 45(ii) a The fermentation culture has a cell Colony Forming Unit (CFU) of 2 × 10 or more9/mL。
In a fourth aspect, the invention provides a lysine bacillus fusiformis preparation for degrading polystyrene plastics according to the second aspect of the invention or a lysine bacillus fusiformis preparation for degrading polystyrene plastics according to the third aspect of the invention.
According to the invention, the application comprises the step of adding the bacillus fusiformis preparation into a degradation culture medium containing the polystyrene plastic to degrade the polystyrene plastic.
Preferably, the polystyrene plastic comprises environmental polystyrene plastic waste; further preferably, the polystyrene plastic comprises environmental polystyrene plastic film waste and polystyrene micro plastic.
In some embodiments of the present invention, the degradation medium is a liquid medium with polystyrene plastic as a sole carbon source, and preferably, the degradation medium comprises the following components in 1L of water, based on 1L of water:
NaHCO3 0.2g；
(NH4)2SO4 1.0g；
CaCO3 0.1g；
Na2HPO4•12H2O 4.37g；
NaH2PO4•2H2O 1.22g；
FeSO4•7H2O 0.01g；
MgSO4•7H2O 0.01g；
CuSO4•5H2O 0.001g；
MnSO4•5H2O 0.001g；
ZnSO4•7H2O 0.001g；
preferably, the pH of the degradation medium is 7.0.
In other embodiments of the invention, the lysinibacillus fusiformis preparation is used in an amount of 0.01g/mL, the degradation treatment temperature is 28-30 ℃, and the degradation treatment time is 4 weeks.
The polystyrene degradation strain-lysine bacillus fusiformis PS-02 strain is obtained by separation and screening, can be effectively used for biodegradation of polystyrene, such as polystyrene plastic membranes, particles and powder, polystyrene plastic degradation is carried out by adopting the strain, obvious biological erosion holes are formed on the surface of the polystyrene membrane after 30 days of treatment, the membrane hydrophilicity is obviously improved, the surface damage is serious, the degradation is green and environment-friendly, the cost is low, the operation is convenient, the strain is suitable for treating polystyrene waste which is difficult to degrade in the natural environment, and the strain has important significance for protecting the ecological environment and the body health of human bodies.
Drawings
For the present invention to be readily understood, the following description is made with reference to the accompanying drawings.
FIG. 1 shows a 16S rDNA-based molecular evolutionary tree of B.fusiformis PS-02.
FIG. 2 shows the growth curve of lysine bacillus fusiformis PS-02.
FIG. 3 shows the results of the particle size distribution of lysine bacillus fusiformis PS-02 for degrading polystyrene micro-plastic samples in water.
FIG. 4 shows the result of IR spectroscopy analysis of a sample of polystyrene micro-plastic in a water body degraded by Lysinibacillus fusiformis PS-02.
FIG. 5 shows the result of SEM analysis of the sample of polystyrene micro-plastic in water degraded by Lys. fusiformis PS-02.
FIG. 6 shows the result of the contact angle analysis of the sample of the polystyrene plastic film in the degradation water body by the lysine bacillus fusiformis PS-02.
FIG. 7 shows the result of SEM analysis of the sample of polystyrene plastic film in water degraded by Lys. fusiformis PS-02.
Strain preservation
The strain is separated and identified by Beijing university of science and technology, has been preserved in China general microbiological culture Collection center (CGMCC for short; China institute of sciences, national institute of microbiology, No. 3, Xilu No. 1, Beijing, the south of the republic of China), and is classified and named as lysine bacillus fusiformis (Lysinibacillus fusiformis)Lysinibacillus fusiformis) The preservation date: 22/11/25/2021, accession No.: CGMCC No. 23975. The strain is named as lysine bacillus fusiformis PS-02 strain (in the invention)Lysinibacillus fusiformis strain PS-02）。
Detailed Description
In order that the invention may be readily understood, a detailed description of the invention is provided below. However, before the invention is described in detail, it is to be understood that this invention is not limited to 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.
Term of
The term "cell" as used herein refers to a live cell and/or a dead cell of a bacterium.
The term "spore" in the invention refers to a dormant body with very strong stress resistance formed by bacillus under certain conditions.
The terms "lysine bacillus fusiformis" and "lysine bacillus fusiformis" or "lysine bacillus fusiformis" as used herein are used interchangeably.
The terms "polystyrene micro-plastic" and "polystyrene nano-micro-plastic" are used interchangeably in this invention.
The "water" used in the culture medium or fermentation culture process of the invention means, without particular designation, sterile pure water obtained by filtration through a 0.22 μm filter membrane.
II, embodiments
As mentioned above, the existing bacteria for degrading polystyrene plastics are not satisfactory, and have the problems of incomplete degradation of polystyrene plastics, low degradation efficiency, incapability of degrading polystyrene micro-plastics and the like. In view of this, the present inventors have conducted extensive studies on biodegradation of polystyrene plastics.
On the basis of long-term waste treatment research, the inventor successfully domesticates and screens a microbial pure strain for efficiently and biologically degrading polystyrene plastics from an environmental plastic waste soil sample, and an enzyme generated by the strain can catalyze and degrade the polystyrene plastics, particularly can efficiently degrade the polystyrene plastics, and has an important application prospect in the aspect of efficiently and biologically degrading the polystyrene plastics.
Accordingly, the present invention relates in a first aspect to a bacillus for the biodegradation of polystyrene plastic.
The inventor successfully screens out a bacillus strain which can generate enzymes for catalyzing and degrading polystyrene plastics and polystyrene micro plastics from an environmental plastic waste soil sample (namely, the surface of the plastic waste in a domestic garbage landfill is contacted with soil). Extracted genomic DNA, amplified by PCR and identified as Bacillus fusiformis by molecular characterization based on 16S rDNA sequencingLysinibacillus fusiformisspBased on the above, this strain was identified and designated lysine bacillus fusiformis PS-02 strain (b.fusiformis-PS-02:)Lysinibacillus fusiformis strain PS-02). The strain is preserved in the China general microbiological culture Collection center, and the preservation number is as follows: CGMCC No. 23975.
The research result shows that the bacterial cells and/or spores of the lysine bacillus fusiformis PS-02 strain with the concentration of 0.01g/mL can reduce the water contact angle of the polystyrene plastic film from 97.0 degrees +/-4.60 degrees to 64.0 degrees +/-3.62 degrees within 4 weeks (see figure 6); meanwhile, the characterization of a Fourier infrared spectrometer shows that the bacterial cells of the lysinibacillus fusiformis PS-02 strain with the concentration of 0.01g/mL can make the polyphenyl in 4 weeksThe infrared spectrum of the ethylene micro plastic is 800-1700 cm in wave number–1New characteristic peaks are generated (see fig. 4), it can be concluded that the degradation of the polystyrene micro-plastic generates active groups such as C = O, C-C, C = C, which indicates that the macromolecular polystyrene is depolymerized or chain-broken and generates new products, and the scanning electron microscope examination shows that the surface of the flat and smooth polystyrene micro-plastic particles is provided with obvious ravines and damages (see fig. 5), which indicates that the polystyrene micro-plastic is significantly degraded.
Based on the above, the second to fourth aspects of the present invention further provide a use or application of the bacillus for biodegradation of polystyrene plastic according to the first aspect of the present invention.
Specifically, the second aspect of the present invention provides a lysine bacillus fusiformis preparation for biodegradation of polystyrene plastics, which belongs to a microorganism preparation for biodegradation of polystyrene plastics, and which contains bacterial cells and/or spores of bacillus according to the first aspect of the present invention.
In some preferred embodiments of the invention, the lysinibacillus fusiformis preparation contains spores of the bacillus as described in the first aspect of the invention.
According to some embodiments of the invention, the formulation of lysine bacillus fusiformis for biodegradable polystyrene plastic is a liquid formulation.
In some embodiments of the invention, the concentration of bacterial cells and/or spores of the Bacillus in the liquid preparation for degrading polystyrene plastic is more than or equal to 1 × 109PermL, more preferably (1-3). times.109PermL, more preferably (2-3). times.109/mL。
According to other embodiments of the present invention, the formulation of lysine bacillus fusiformis for biodegradable polystyrene plastic is a solid powder formulation.
In some embodiments of the invention, the polystyrene plastic-degrading lysinibacillus fusiformis formulation is a solid powder formulation; preferably, in the solid powder preparation for degrading polystyrene plastics, the bacterial cells of the bacillus and/orThe content of spore is more than or equal to 1 × 1011(1-3). times.10 is more preferable11(2-3). times.10 is more preferable11/g。
In a third aspect of the present invention, there is provided a method for preparing a bacillus lysinibacillus fusiformis preparation for biodegradable polystyrene plastic according to the second aspect of the present invention, comprising:
b, inoculating the fermentation strain into a fermentation culture medium for fermentation culture to obtain a fermentation culture of bacillus;
step C, carrying out centrifugal separation treatment on the fermentation culture of the bacillus, and harvesting bacillus wet thalli containing bacterial cells and/or spores to be used as a fusiform lysine bacillus preparation;
wherein the fermentation strain is obtained by seed culture of corresponding strains.
As known to those skilled in the art, the 16S rRNA is currently used internationally for molecular identification of bacteria, and thus, 16S rRNA can be used for alignment to obtain homology in similarity comparison. A phylogenetic tree is constructed according to the sequencing result of the 16SrDNA, and the sequence comparison result and the phylogenetic tree analysis result show that: the degrading bacteria PS-02 is most similar to the fusiform lysine bacillus JQ900544.1 (the similarity of the sequence is 99 percent), and the bacterial strain is the fusiform lysine bacillus (Lysinibacillus fusiformis). FIG. 1 shows a molecular evolutionary tree based on 16S rDNA, the Bacillus of the present invention being the Bacillus fusiformis PS-02 strain. Therefore, the fermentation strain used in the present invention is not limited to the field isolate used in the present invention, and 16S rDNA is a DNA sequence corresponding to the coding rRNA on the chromosome of the bacterium and exists in the chromosomal genome of all bacteria.
Based on the above it will be readily appreciated that in the present invention, the corresponding strain of the species fermentative is the Bacillus strain of the first aspect of the present invention. That is, on the premise of not changing the 16S rDNA of the lysine bacillus fusiformis PS-02, a person skilled in the art can obtain a strain highly homologous to the 16S rDNA of the lysine bacillus fusiformis PS-02 by simply screening or mutagenizing the lysine bacillus fusiformis PS-02 of the invention, and obtain a strain having the same or similar function of biodegradation of polystyrene plastics.
In the step C, the centrifugal separation treatment includes subjecting the liquid fermentation culture to centrifugal separation to obtain a precipitate (i.e., bacterial cells and/or spores of bacillus), resuspending and washing the precipitate with physiological saline, and then subjecting the precipitate to centrifugal separation to obtain bacterial cells and/or spores of bacillus.
The conditions for the centrifugation in the step C are not particularly limited in the present invention, and in some embodiments of the present invention, the substance to be separated may be centrifuged for 10-20 min at 8000-.
According to the method, the fermentation culture is shaking table or fermentation tank fermentation culture of strains, and the fermentation strains are inoculated into a fermentation culture medium in the form of seed liquid. The inoculation amount of the seed liquid is 0.1-1% (v/v); preferably, the inoculation amount of the seed liquid is 0.2-0.5% (v/v); further preferably, the amount of the seed liquid to be inoculated is 0.2% (v/v). In the seed liquid, the Colony Forming Unit (CFU) of the strain is (1-3). times.109/mL。
Specifically, the fermentation medium comprises the following components in 1L of water in terms of 1L of water:
20-30g of monohydrate glucose;
1-3g of malt extract powder;
KH2PO4 1-3g；
MgSO4•7H2O 1-3g；
MgCl2•4H2O 0.2-0.6g；
CaCl2 0.5-1.5g；
5-10g of sodium chloride;
15-25g of beef extract;
5-10g of peptone;
5-10g of yeast extract;
in order to obtain higher yield of wet thalli of the lysine bacillus fusiformis PS-02 strain, optimization research is carried out on a fermentation culture medium, and the result shows that the fermentation culture medium is prepared according to the following components and is favorable for fermentation culture, and the fermentation culture medium comprises the following components in 1L of water:
20g of dextrose monohydrate;
1g of malt extract powder;
KH2PO4 1g；
MgSO4•7H2O 1g；
MgCl2•4H2O 0.2g；
CaCl2 0.5g；
5g of sodium chloride;
15g of beef extract;
5g of peptone;
5g of yeast extract;
in some embodiments of the invention, the initial pH of the fermentation medium is adjusted with 40% (wt/v) sodium hydroxide solution and 36% (v/v) hydrochloric acid solution, and the pH of the fermentation medium is 6.5 to 7.5, preferably 6.8 to 7.2, and more preferably 7.2.
According to some embodiments of the present invention, the method for preparing a bacillus according to the present invention further comprises step a: the lysine bacillus fusiformis PS-02 strain monoclonal colony provided by the invention is selected and inoculated into 100mL of fermentation liquid culture medium, and after shake cultivation is carried out for 2 days at the temperature of 35 ℃ and the rotating speed of 200r/min, a fermentation strain (seed solution) is prepared.
The inventors studied the effect of different temperatures on the growth of lysine bacillus fusiformis PS-02 and found that lysine bacillus fusiformis PS-02 grew fast at a temperature of 30-35 c, preferably at 35 c.
According to the invention, the preparation method also comprises a step D after the step C, and the solid powder preparation for degrading the polystyrene plastic is obtained after the bacillus wet thalli containing the bacterial cells and/or the spores are freeze-dried.
In the above step B, theThe optical density (OD 680 nm) of the fermentation culture is more than or equal to 45; the fermentation culture has a cell Colony Forming Unit (CFU) of 2 × 10 or more9/mL。
The fourth aspect of the present invention provides a use of the lysenin bacillus fusiformis preparation for biodegradable polystyrene plastic according to the second aspect of the present invention or the lysenin bacillus fusiformis preparation for biodegradable polystyrene plastic prepared by the preparation method according to the third aspect of the present invention in degradation of polystyrene plastic, which can be understood as a method for degrading polystyrene plastic using the lysenin bacillus fusiformis preparation for biodegradable polystyrene plastic according to the second aspect of the present invention or the lysenin bacillus fusiformis preparation for biodegradable polystyrene plastic prepared by the preparation method according to the third aspect of the present invention.
In the invention, the polystyrene plastic comprises environmental polystyrene plastic waste; further preferably, the polystyrene plastic comprises environmental polystyrene plastic film waste and polystyrene micro plastic.
According to the invention, the application comprises the step of adding the bacillus fusiformis preparation into a degradation culture medium containing the polystyrene plastic to degrade the polystyrene plastic.
In some specific embodiments of the present invention, the degradation medium is a liquid medium with polystyrene plastic as a sole carbon source, and preferably, the degradation medium comprises the following components in 1L of water, based on 1L of water:
NaHCO3 0.2g；
(NH4)2SO4 1.0g；
CaCO3 0.1g；
Na2HPO4•12H2O 4.37g；
NaH2PO4•2H2O 1.22g；
FeSO4•7H2O 0.01g；
MgSO4•7H2O 0.01g；
CuSO4•5H2O 0.001g；
MnSO4•5H2O 0.001g；
ZnSO4•7H2O 0.001g；
preferably, the pH of the degradation medium is 7.0.
Further preferably, the dosage of the lysinibacillus fusiformis preparation is 0.01g/mL, the degradation treatment temperature is 28-30 ℃, and the degradation treatment time is more than or equal to 4 weeks.
III, correlation detection method in the invention
(1) The cell and/or spore concentration of the present invention is determined by the following method:
the method comprises the steps of taking a culture of the lysine bacillus fusiformis PS-02, diluting the culture by a certain multiple of physiological saline, and directly measuring the concentration of cells and/or spores in the culture by a flow cytometer (SYSMEX, Germany).
(2) The concentration of the crude enzyme protein is determined by the following method:
taking a cell-free extracting solution of the fusiform lysine bacillus PS-02, diluting the cell-free extracting solution by a phosphate buffer solution by a certain multiple, adding a Coomassie brilliant blue G-250 dye reagent in proportion, reacting for 10 minutes, measuring absorbance at 595nm by using a 722S visible spectrophotometer (Shanghai prismatic light), and calculating the protein concentration by adopting a standard curve method.
III example
The present invention will be specifically described below with reference to specific examples. The experimental methods described below are, unless otherwise specified, all routine laboratory procedures. The experimental materials described below, unless otherwise specified, are commercially available.
(1) In the following examples, the composition of the fermentation broth of lysine bacillus fusiformis PS-02 was (per liter): 20g of dextrose monohydrate, 1g of malt extract powder and KH2PO4 1g，MgSO4•7H2O 1g，MgCl2•4H2O 0.2g，CaCl20.5g, 5g of sodium chloride, 15g of beef extract, 5g of peptone and 5g of yeast extract, and the initial pH value of the fermentation medium is adjusted to 7.2 by using 40% (wt/v) of sodium hydroxide solution and 36% (v/v) of hydrochloric acid solution. 100ml of the prepared growth medium was added to a 500 ml Erlenmeyer flask, sterilized at high temperature and high pressure (121 ℃) for 20 minutes, and then sterilized again under ultraviolet irradiation in a clean bench for 20 minutes.
(2) In the following examples, the composition of the inorganic salt liquid medium (pH =7.0) used for degradation treatment was (per liter):
NaHCO3 0.2g；
(NH4)2SO4 1.0g；
CaCO3 0.1g；
Na2HPO4•12H2O 4.37g；
NaH2PO4•2H2O 1.22g；
FeSO4•7H2O 0.01g；
MgSO4•7H2O 0.01g；
CuSO4•5H2O 0.001g；
MnSO4•5H2O 0.001g；
ZnSO4•7H2O 0.001g。
example 1: preparation of lysine bacillus fusiformis liquid preparation
(1) Preparing a seed solution: selecting a single colony of the lysinibacillus fusiformis PS-02, inoculating the single colony into 100ml of fermentation liquid culture medium, performing shake culture at 35 ℃ and 200r/min for 2d to obtain a fermentation strain (seed liquid), wherein the Colony Forming Unit (CFU) of the strain in the seed liquid is 3 multiplied by 109/mL。
(2) And (3) carrying out fermentation culture on lysine bacillus fusiformis:
inoculating 0.2% (v/v) lysine bacillus fusiformis PS-02 bacterial liquid into a triangular flask liquid culture medium under the aseptic condition in a clean workbench, carrying out fermentation culture in a shaking table fermentation culture mode of strain dissociation, inoculating a fermentation strain into the fermentation culture medium in a seed liquid mode, carrying out batch culture for 2 days at the temperature of 35 ℃ and the rotation speed of the shaking table of 200r/min, and obtaining a fermentation culture;
the Optical Density (OD) of the culture was measured every 6 hours during the fermentation680nm) 3 times per measurement and averaging, the growth curve of the degrading bacteria was plotted (see FIG. 2), and the Optical Density (OD) of the fermentation culture after 48 hours of fermentation680nm) Not less than 50; the cell Colony Forming Unit (CFU) of the culture is not less than 3 × 109and/mL. And separating the wet thallus obtained from the fermentation culture to prepare the microbial inoculum.
(3) Cell separation:
the lysine bacillus fusiformis PS-02 cells and/or spores were harvested as a liquid preparation of lysine bacillus fusiformis by decanting the supernatant after centrifugation (8000 rpm, 10 minutes).
Example 2: application of polystyrene micro-plastic in water body in degradation
(1) Degradation treatment
50mL of inorganic salt liquid culture medium (without carbon source) and ultraviolet sterilized polystyrene micro plastic (1.0 g) with the particle size of 80nm or 100 mu m are added into a 200mL conical flask to form the inorganic salt liquid culture medium with the polystyrene plastic as the only carbon source. 0.5g of plastic degradation microbial inoculum obtained by fermentation production is added into the conical flask, the conical flask is placed in a shaking table at 35 ℃ and 200r/min for culture, and degradation performance detection is carried out after 4 weeks.
(2) Sample treatment: the collected liquid culture medium is stirred and washed for 2 hours by using 2.0 percent Sodium Dodecyl Sulfate (SDS) water solution, bacterial colonies attached to the micro-plastic are washed off, and the liquid culture medium is dried in an oven at 65 ℃ after being washed by clear water.
(3) Measuring the granularity of a sample:
particle size analyzer: the working principle of the laser particle sizer is Dynamic Light Scattering (DLS), which is a physical characterization method used for measuring the particle size distribution in a solution or suspension, and can also be used for measuring the behavior of complex fluids such as a concentrated polymer solution. By illuminating the particles with laser light, the scattering intensity at a particular angle will fluctuate over time and can be detected using a sensitive Avalanche Photodiode Detector (APD). The detection is carried out on a Zetasizer Nano ZS90 laser particle size analyzer with the measurement range of 20 nm-900 nm by using Dynamic Light Scattering (DLS). The intensity variations are analyzed with a digital autocorrelator, which generates a correlation function. The curves can be analysed to give the size and size distribution, see figure 3.
The detection result of the laser particle size analyzer shows that the micro plastic is significantly agglomerated under the action of the degrading bacteria, the particles of 350 nm are agglomerated from the first 80nm, and considering that the particle size of the bacteria is about micron level, the particles of 350 nm are not bacteria, but are micro plastic particles agglomerated under the action of enzyme and protein secreted by the bacteria, so that the degrading bacteria can be deduced to agglomerate and adhere the small micro plastic together by secreting the related enzyme or protein, the dispersion of the micro plastic in the water body environment is reduced, the adsorption, enrichment and degradation effects are achieved, and the micro plastic can be applied to the biological purification carrier of the water body.
(4) Fourier transform infrared spectroscopy (FTIR) analysis:
the samples were measured using fourier transform infrared spectroscopy (FTIR) on a Nicolet iS50 FTIR spectrometer. The recording range of the spectrum is 400-4000 cm–1At least 16 times of scanning, and the spectral resolution is 0.48cm–1. Peaks were identified using OMNIC software. And the metabolite content was determined based on the corresponding number of peaks and peak area, and the results are shown in FIG. 4.
The characterization result of the Fourier infrared spectrometer shows that the functional group of the micro-plastic treated by the degrading microbial inoculum is obviously changed, and compared with a control group, the product treated by the microbial inoculum has the wave number of 800-1700 cm–1New characteristic peaks are generated, and the conclusion that the plastic is degraded to generate active groups such as C = O, C-C, C = C can be drawn. Therefore, the polystyrene is obviously degraded under the treatment of the microbial inoculum, the macromolecular polymer is depolymerized or chain-broken, and a new product is generated.
(5) Scanning Electron Microscope (SEM):
and selecting a small amount of samples, performing brittle fracture by using liquid nitrogen, fixing the samples on a metal sample table by using double-sided adhesive tape, placing the fixed samples in a vacuum evaporator, vacuumizing and spraying gold for 25s, and improving the conductivity and secondary electron yield of the samples. The sample was placed in a scanning electron microscope, observed with an accelerating voltage of 2KV, taken at different magnifications at 360 °, and a representative microstructure photograph was taken, as shown in fig. 5.
From the detection result of the scanning electron microscope, it can be known that, after the microbial inoculum of the polystyrene plastic degrading bacteria is used for treating the polystyrene micro plastic particles, obvious gully and breakage appear on the surfaces of the micro plastic particles, while the surfaces of the micro plastic particles of the control group are smoother. The microbial inoculum can grow by taking polystyrene as a unique carbon source in the growth process, and the surface structure of plastic particles is damaged by eroding the plastic particles, so that the microbial inoculum can generate obvious degradation effect on micro plastic particles.
Example 3: degradation application of polystyrene plastic membrane in water body
(1) Degradation treatment
Degradation application: 50mL of inorganic salt liquid culture medium (without carbon source) and a plurality of polystyrene thin film sheets (1.0 g) subjected to ultraviolet sterilization treatment are added into a 200mL conical flask to form the inorganic salt liquid culture medium taking polystyrene plastic as the only carbon source. 0.5g of plastic degradation microbial inoculum obtained by fermentation production is added into the conical flask, the conical flask is placed in a shaking table at 35 ℃ and 200r/min for culture, and degradation performance detection is carried out after 4 weeks.
(2) Hydrophobic contact angle determination:
and (3) stirring and washing the collected polystyrene plastic membrane for 2 hours by using a 2.0% Sodium Dodecyl Sulfate (SDS) water solution, washing to remove bacterial colonies attached to the plastic membrane, washing by using clear water, and drying in an oven at 65 ℃. The OCA20 optical contact angle tester measures the contact angle of water drop on the surface of micro plastic. In the measurement, the particles were dropped on a slide glass, and the particles were dried by heating, and the amount of the drop was 5. mu.L, and 5 different points were measured for each sample, and the average value was obtained, as shown in FIG. 6.
According to the water contact angle experiment result, after the polystyrene plastic film is treated by the microbial inoculum of the polystyrene plastic degrading bacteria, the water contact angle on the surface of the film is reduced from 97.0 degrees +/-4.60 degrees to 64.0 degrees +/-3.62 degrees, the hydrophobicity on the surface of the plastic is obviously reduced, and the hydrophilicity is improved, so that the high-efficiency degrading capability of the degrading bacteria on the polystyrene plastic is further demonstrated, the hydrophobicity of the plastic can be reduced, and the biodegradation of the plastic is accelerated.
(3) Elemental analysis:
and (3) stirring and washing the collected polystyrene plastic membrane for 2 hours by using a 2.0% Sodium Dodecyl Sulfate (SDS) water solution, washing to remove bacterial colonies attached to the plastic membrane, washing by using clear water, and drying in an oven at 65 ℃. Using an einlemond tower UNICUBE elemental analyzer, CHNS mode: combustion of sample in pure oxygen to CO2、H2O、N2And SO2The content of C, H, N, S in the sample is measured by thermal conductivity detection after the separation by the chromatographic column, and the result is as follows:
group N (%) C (%) H (%);
theoretical value 092.37.69;
control group 091.97.37;
experimental group 086.57.55;
the element analysis and test results show that after the polystyrene plastic film is treated by the microbial inoculum of the polystyrene plastic degrading bacteria, compared with a control group, the content of the C element in the treated plastic is obviously reduced by 5.4 percent, which indicates that the microbial inoculum can grow by taking polystyrene as a unique carbon source in the growth process, and the reduced C element can be decomposed into CO under the metabolic action of the degrading bacteria2The dissipation shows that the degrading bacteria can generate obvious degradation effect on the polystyrene plastic film.
(4) Scanning Electron Microscope (SEM):
and (3) stirring and washing the collected polystyrene plastic membrane for 0.5h by using a 2.0% Sodium Dodecyl Sulfate (SDS) water solution, washing out the culture medium attached to the plastic membrane, washing by using clear water, and then placing in a 2.5% glutaraldehyde solution for storage. And selecting a small amount of samples, performing brittle fracture by using liquid nitrogen, fixing the samples on a metal sample table by using double-sided adhesive tape, placing the fixed samples in a vacuum evaporator, vacuumizing and spraying gold for 25s, and improving the conductivity and secondary electron yield of the samples. The sample was placed in a scanning electron microscope, observed with an accelerating voltage of 2KV, taken at different magnifications at 360 °, and a representative microstructure photograph was taken, as shown in fig. 7.
According to the detection result of the scanning electron microscope, after the polystyrene plastic film is treated by the microbial inoculum of the polystyrene plastic degrading bacteria, the degrading bacteria have obvious biotransformation effect on the plastic film, the surface of the plastic film is obviously crushed at multiple positions, a large amount of bacteria are attached to the crushed positions, and the bacteria grow well, so that the microbial inoculum can grow by taking polystyrene as a unique carbon source in the growth process, and the surface structure of the plastic film is damaged by eroding plastic particles, so that the polystyrene plastic film is obviously degraded.
It should be noted that the above-mentioned embodiments are only for explaining the present invention, and do not constitute any limitation to the present invention. The present invention has been described with reference to exemplary embodiments, but the words which have been used herein are words of description and illustration, rather than words of limitation. The invention can be modified, as prescribed, within the scope of the claims and without departing from the scope and spirit of the invention. Although the invention has been described herein with reference to particular means, materials and embodiments, the invention is not intended to be limited to the particulars disclosed herein, but rather extends to all other methods and applications having the same functionality.
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