CN115960759B - Polystyrene plastic and polyethylene plastic degrading bacterium, screening method and application thereof - Google Patents
Polystyrene plastic and polyethylene plastic degrading bacterium, screening method and application thereof Download PDFInfo
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Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
- Y02W30/00—Technologies for solid waste management
- Y02W30/50—Reuse, recycling or recovery technologies
- Y02W30/62—Plastics recycling; Rubber recycling
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a Polystyrene (PS) plastic and Polyethylene (PE) plastic degrading strain, which is identified as ester-flavor bacillus (Microbacteriumesteraromaticum) SW3 and is preserved in China general microbiological culture Collection center (China Committee) for culture Collection of microorganisms, wherein the preservation number is: CGMCC No. 25677; the preservation time is as follows: 2022, 9 and 9. The strain has remarkable degradation efficiency on PS plastics and PE plastics, such as PS plastic cutlery boxes, PE agricultural mulching films and the like, provides strain resources for biodegradation of PS plastics and PE plastics, and has good application prospect and ecological environmental protection value.
Description
Technical Field
The invention relates to the field of biodegradation of plastic pollution, in particular to a strain of polystyrene (Polystyrene, PS) plastic and Polyethylene (PE) plastic degrading bacteria, a screening method thereof and application of the strain in biodegradation of PS plastic and PE plastic polluted samples.
Background
PS and PE are synthetic organic high polymers, and the products have the advantages of portability, durability, insulation, plasticity and the like, and meanwhile, the cost is low, so that the products are widely used in the world. PS is often used for preparing foamed plastic and disposable tableware, preserving heat and packaging in buildings, and PE is often used for preparing disposable plastic bags, preservative films, agricultural mulching films, and the like. A large number of PS, PE related articles accumulate in the environment, posing a serious threat to the global ecosystem.
The traditional plastic pollution treatment methods include landfill, incineration, thermal cracking, secondary processing and the like, but the treatment methods have high cost, high energy consumption and easy secondary pollution to the environment. The microbial degradation of plastic pollution has the advantages of green, environmental protection, friendly property, low cost and the like, and has great application potential. However, the existing PS plastics and PE plastics degrading bacteria have limited bacterial strain resources, low degradation efficiency and main degradation of single plastic components, which prevent the application of the PS plastics and PE plastics degrading bacteria.
Disclosure of Invention
The invention provides a strain with remarkable degradation efficiency on PS plastics and PE plastics, a separation and screening method thereof and application of the strain in biodegradation of PS plastics and PE plastics polluted samples.
The invention provides a PS plastic and PE plastic degrading strain which is a micro bacillus esteranus SW3 (Microbacterium esteraromaticum SW 3) and is preserved in the China general microbiological culture Collection center with the preservation address of: the institute of microorganisms at national academy of sciences of China, national academy of sciences, no. 1, north Star West way, no.3, chat.Chao, beijing, city; the preservation number is: CGMCC No. 25677; the preservation time is as follows: 2022, 9 and 9.
Preferably, the 16S rDNA sequence of the strain SW3 is shown in a sequence table SEQ ID NO: 1.
The invention provides a separation and screening method of PS plastic and PE plastic degrading bacteria strains, which comprises the following steps:
(1) Soil samples polluted by PS and PE plastics for a long time are used as strain sources, enrichment culture is carried out in an inorganic salt culture medium which takes PS as a sole carbon source, and PS powder is sterilized before ultraviolet rays. Observing the state of the culture medium every 24 hours, culturing 20 mu L of culture solution in 100mL of fresh inorganic salt culture medium containing 0.1% PS for 5 times when the culture medium is obviously turbid;
(2) Diluting the culture solution obtained by continuous enrichment culture, separating by using a streaking method to obtain PS degrading bacteria, and numbering and storing;
(3) Culturing the obtained PS degrading bacteria in an inorganic salt culture medium with PE as a unique carbon source, and enriching to obtain strains capable of degrading PE simultaneously, thereby obtaining degrading bacteria SW3 capable of simultaneously utilizing PS and PE.
The inorganic salt culture medium comprises ：K2HPO4 0.7g,KH2PO4 0.7g,MgSO4·7H2O 0.7g,NH4NO31.0g,NaCl 0.005g,FeSO4·7H2O 0.002g,ZnSO4·7H2O 0.002g,MnSO4·H2O 0.001g, of dissolving the reagent in 1L of deionized water, and regulating the pH to 7.2.
The invention relates to a PS and PE degrading bacterium, which has colony morphological characteristics that: the colony is milky white, round in shape, micro-raised, transparent, smooth and moist in surface and regular in edge. Gram staining was purple and positive. The physiological and biochemical characteristics show that the enzyme can utilize lactose, sucrose and starch, does not generate hydrogen sulfide, and has positive contact enzyme and negative cytochrome C oxidase. The sequences of the strain 16S rDNA genes of SW3 are amplified and sequenced, and BLAST comparison analysis is carried out on the obtained sequences and the existing sequences in NCBI database, so that the results show that the sequence consistency of the SW3 and the strains of the multi-strain ester micro-bacillus is over 99 percent, the evolutionary distance is short, and the strains are identified to be ester micro-bacillus (Microbacterium esteraromaticum) by combining the physiological and biochemical characteristics of the strains.
The degrading bacteria SW3 provided by the invention are separated from plastic garbage accumulation positions (N29 DEG 49', E106 DEG 26') around the bamboo forest.
The degrading bacteria SW3 provided by the invention has remarkable degradation efficiency on PS plastics and PE plastics, and is characterized in that: SW3 is inoculated to a PS or PE product, and from the 21 st day of culture, a large amount of bacteria are adhered to the surface of the product and are observed under an optical microscope and a scanning electron microscope, so that a large amount of damages appear; the Fourier infrared spectrum detection shows that carbonyl and hydroxyl functional groups are generated on the surface of the PS product, and the two peak intensities of the main alkane groups in the PE product polyethylene skeleton are reduced and degraded.
The invention has the following advantages:
(1) By adopting the separation and screening method of the plastic degrading bacteria, strain resources capable of degrading PS and PE simultaneously can be obtained.
(2) The strain obtained by the invention has remarkable effect on the biodegradation of PS and PE plastic products.
(3) The strain obtained by the invention can be used for biodegradation of samples polluted by PS or PE plastics, and can also be used for biodegradation of samples polluted by PS and PE plastics at the same time.
(4) The invention can provide new strain resources and new ideas for biological treatment of PS and PE plastics in the environment, and has good application prospect.
Drawings
In order to make the objects, technical solutions and advantageous effects of the present invention more clear, the present invention provides the following drawings for description:
FIG. 1 is a colony morphology feature diagram of the PS plastic and PE plastic degrading bacteria ester micro-bacillus SW3 provided by the invention.
FIG. 2 is a graph showing the growth of the degrading bacteria SW3 in an inorganic salt culture medium with PS or PE as the only carbon source.
FIG. 3 shows a phylogenetic tree of the degrading bacterium SW3 based on the 16S rDNA gene sequence analysis.
FIG. 4 is a diagram showing the morphology observation of PS cutlery box and PE preservative film under an optical microscope and a scanning electron microscope after the degradation bacteria SW3 provided by the invention are inoculated into an inorganic salt culture medium taking the PS cutlery box or the PE preservative film as the only carbon source and cultured for 21 days at 30 ℃ and 150 r/min.
FIG. 5 shows the Fourier infrared spectra of the PS cutlery box and PE preservative film after the degrading bacteria SW3 provided by the invention are inoculated into an inorganic salt culture medium taking the PS cutlery box or the PE preservative film as the only carbon source and cultured for 21 days at the temperature of 30 ℃ and at the speed of 150 r/min.
Detailed Description
In order to clearly and completely describe the specific implementation method of the present invention and the technical solution in the embodiments of the present invention, the following description will be made with reference to the accompanying drawings in the embodiments of the present invention. The embodiments described below are intended to be illustrative of some, but not all, of the embodiments of the present invention and all embodiments that may be made by one of ordinary skill in the art without undue burden are within the scope of the present invention.
The invention is further illustrated by the following examples:
Example 1
The invention provides a strain of PS plastic and PE plastic degrading bacteria SW3, which has colony morphological characteristics of milky colony, round shape, micro-bulge, transparency, smooth surface, wetness and regular edge on an LB plate, as shown in figure 1. The strain belongs to micro bacillus esteranus (Microbacterium esteraromaticum), is preserved in China general microbiological culture Collection center (China Committee) for culture Collection of microorganisms, and has the preservation address: the institute of microorganisms at national academy of sciences of China, national academy of sciences, no. 1, north Star West way, no. 3, chat.Chao, beijing, city; the preservation number is: CGMCC No. 25677; the preservation time is as follows: 2022, 9 and 9.
Example 2
The invention provides a separation and screening method of PS plastic and PE plastic degrading bacteria, which specifically comprises the following steps:
(1) Taking soil samples polluted by PS and PE plastics for a long time as strain sources, weighing 1g of the collected soil samples, uniformly mixing the soil samples with 100mL of sterile water, and fully shaking the mixture to prepare the soil diluent. 20. Mu.L of the soil dilution was taken in 100mL of an inorganic salt medium containing 0.1% PS to isolate PS degrading bacteria, and PS powder was sterilized prior to ultraviolet irradiation. Meanwhile, 20 mu L of soil dilution is inoculated into 100mL of inorganic salt culture medium without carbon source for comparison, and the culture is simultaneously carried out at 30 ℃ and 150 r/min. Every 24 hours, when the culture medium appears obviously turbid, 20 mu L of culture solution is taken to be cultured in 100mL of fresh inorganic salt culture medium containing 0.1% PS, and the culture is repeated for 5 times.
(2) Diluting the culture solution obtained by continuous enrichment culture, separating by using a streaking method to obtain PS degrading bacteria, and numbering and storing;
(3) Culturing the obtained PS degrading bacteria in an inorganic salt culture medium with PE as a unique carbon source, and enriching to obtain strains capable of degrading PE simultaneously, thereby obtaining degrading bacteria SW3 capable of simultaneously utilizing PS and PE.
The inorganic salt medium used in the examples of the present invention had a composition ：K2HPO4 0.7g,KH2PO4 0.7g,MgSO4·7H2O0.7g,NH4NO3 1.0g,NaCl 0.005g,FeSO4·7H2O 0.002g,ZnSO4·7H2O 0.002g,MnSO4·H2O 0.001g,. The above-mentioned reagents were dissolved in 1L of deionized water, and the pH was adjusted to 7.2.
Example 3
Identification of strains
1. Other identification: gram staining of the strain SW3 thalli to purple indicates that the strain belongs to gram-positive bacteria; the strain SW3 strain is subjected to physiological and biochemical characteristic measurement, and can be found to utilize lactose, sucrose and starch, hydrogen sulfide is not generated, and meanwhile, the contact enzyme is positive, and the cytochrome C oxidase is negative.
2. Determination of the growth curve: 20. Mu.L of the purified bacterial liquid was inoculated into 100mL of an inorganic salt medium containing 2% PS or PE, and simultaneously inoculated into an inorganic salt medium without a carbon source as a control, and cultured at 30℃at 150 r/min. Every 24 hours, the plastic powder in the culture medium was filtered with filter paper and the OD 600 of the filtrate was measured, and the growth curve thereof was plotted as shown in fig. 2. The results showed that SW3 was brought into the logarithmic growth phase at 48h of inoculation in a medium containing 2% PS or inorganic salts of PS, with the 96h bacterial suspension concentration reaching the maximum.
3. Amplification and sequencing of the 16S rDNA Gene sequence
(1) 16S rDNA Gene sequence amplification
The bacterial strain SW3 16S rDNA gene is directly amplified by colony PCR method, the primers used for amplification are 16SrDNA-F1, the base sequence is 5'-AGAGTTTGATCCTGGCTCAG-3',20bp (shown as SEQ ID NO: 2) and 16S rDNA-R1, the base sequence is 5'-TACGGCTACCTTGTTACGACTT-3',22bp (shown as SEQ ID NO: 3).
PCR amplification System (20. Mu.L): the trace amount of the template bacteria, 10 mu L, ddH O9 mu L of Taq enzyme, 0.5 mu L of primer F and 0.5 mu L of primer R.
The PCR reaction conditions were: 3min at 95 ℃;95 ℃ for 30s,60 ℃ for 30s,72 ℃ for 90s,30 cycles; 72 ℃ for 10min; 10min at 4 ℃.
(2) Sequencing of amplified product of 16S rDNA gene sequence
And (3) carrying out electrophoresis detection on the amplified gene product, then sending the amplified gene product to Huada gene sequencing, carrying out BLAST (Basic LocalAlignment Search Tool) comparison analysis on the obtained sequence and the existing sequence in the NCBI database, and carrying out phylogenetic analysis by adopting MEGA X software. The results showed that SW3 was 99% or more identical to the sequences of the Microbacterium esteraromaticum strain and relatively close in evolutionary distance, as shown in FIG. 3. And identifying the SW3 strain as the micro bacillus esteranus SW3 by combining colony morphology, individual morphology staining characteristics, physiological and biochemical characteristics and 16S rDNA phylogenetic analysis.
Example 4
Degradation of PS cutlery box and PE plastic wrap by degrading bacteria SW3
The degrading bacteria SW3 are inoculated into an inorganic salt culture medium which takes a PS cutlery box (2 cm multiplied by 2 cm) or a PE preservative film (2 cm multiplied by 2 cm) as a unique carbon source, the control group is not inoculated with bacteria, shake culture is carried out in a shaking table at 30 ℃ and 150r/min, and the degradation efficiency is observed and detected by methods such as a light mirror, a scanning electron microscope, a Fourier infrared spectrum and the like.
(1) The PS cutlery box, PE plastic wrap optical microscope observe sample treatment and observation method: the plastic products of the treatment group are dyed for 2-3 minutes by using crystal violet dye liquor, after being washed by distilled water, the plastic products are placed in a incubator at 30 ℃, after being dried, a plurality of clean glass slides are taken, distilled water is respectively dripped into the clean glass slides, the plastic products are clamped by forceps and placed on clean water of the glass slides, the plastic products are flattened, and degradation conditions of the surfaces of the plastic products are observed under a microscope (OLYMPUS BX53, japan) and photographed. The control group was observed and photographed after cleaning and drying the tablets in the same manner (fig. 4).
(2) The sample treatment and observation method for the PS cutlery box and the PE preservative film by using a scanning electron microscope comprises the following steps: taking out the cultured plastic product, washing with PBS buffer solution for 3 times, and air drying. Fixing with glutaraldehyde solution with volume fraction of 2.5% for 2h, washing with sterile PBS buffer solution for 3 times, putting the sample into glutaraldehyde solution with volume fraction of 3.0% as soon as possible, fixing for 6h at 4deg.C, rinsing with sterile PBS buffer solution for 3 times, 20min each time. Sequentially treating with 30%, 50%, 70%, 80%, 95% and 100% ethanol solutions at 4deg.C for 20min, removing the sucked solution, treating with acetone at 4deg.C for 20min, and repeating for 2 times. And placing the treated sample in a culture dish for natural drying. And fixing the dried sample on a sample table by using conductive adhesive. And spraying a layer of noble metal with the thickness of about 10nm on the surface of the non-conductive sample. The samples after the metal spraying were subjected to observation photographing under a scanning electron microscope (ZEISS EVO LS10, germany) (fig. 4).
(3) The processing method for detecting the PS cutlery box and PE preservative film sample by the Fourier infrared spectrometer comprises the following steps: the sample was washed with sterile water, then shaken in an ultrasonic cleaner for 30min, soaked in 75% alcohol for 30min, finally washed with sterile water, naturally dried, and the resulting infrared spectrum was further analyzed for functional groups by scanning the wavelength range of 400-4000cm -1 under a fourier transform infrared spectrometer (BRUKER TENSOR 27010409, germany) using attenuated total reflection fourier transform infrared spectroscopy (ATR-FTIR) (fig. 5).
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.
Claims (3)
1. A PS plastic and PE plastic degrading bacterium strain is characterized in that: the PS plastic and PE plastic degrading bacteria are micro bacillus esteranus (Microbacterium esteraromaticum) SW3, and the 16S rDNA sequence of the micro bacillus esteranus is shown in a sequence table SEQ ID NO: 1. Preserving in China general microbiological culture Collection center with a preservation number CGMCC No. 25677; the preservation time is as follows: 2022, 9 and 9.
2. The use of PS plastic, PE plastic degrading bacterial strains according to claim 1, characterized in that: the application of the degradation strain SW3 in biodegradation of samples polluted by PS plastics or PE plastics.
3. The use of PS plastic, PE plastic degrading bacterial strains according to claim 1, characterized in that: the application of the degradation strain SW3 in the biodegradation of samples polluted by PS plastic and PE plastic at the same time.
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