CN110257310B - A kind of vinyon efficient degrading bacteria and its separating screening method and application - Google Patents

A kind of vinyon efficient degrading bacteria and its separating screening method and application Download PDF

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CN110257310B
CN110257310B CN201910743229.7A CN201910743229A CN110257310B CN 110257310 B CN110257310 B CN 110257310B CN 201910743229 A CN201910743229 A CN 201910743229A CN 110257310 B CN110257310 B CN 110257310B
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vinyon
polyethylene
bacterial strain
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efficient degrading
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CN110257310A (en
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涂晨
刘颖
周倩
骆永明
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Yantai Institute of Coastal Zone Research of CAS
Institute of Soil Science of CAS
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    • A—HUMAN NECESSITIES
    • A62—LIFE-SAVING; FIRE-FIGHTING
    • A62D—CHEMICAL MEANS FOR EXTINGUISHING FIRES OR FOR COMBATING OR PROTECTING AGAINST HARMFUL CHEMICAL AGENTS; CHEMICAL MATERIALS FOR USE IN BREATHING APPARATUS
    • A62D3/00—Processes for making harmful chemical substances harmless or less harmful, by effecting a chemical change in the substances
    • A62D3/02—Processes for making harmful chemical substances harmless or less harmful, by effecting a chemical change in the substances by biological methods, i.e. processes using enzymes or microorganisms
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F3/00—Biological treatment of water, waste water, or sewage
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    • A—HUMAN NECESSITIES
    • A62—LIFE-SAVING; FIRE-FIGHTING
    • A62D—CHEMICAL MEANS FOR EXTINGUISHING FIRES OR FOR COMBATING OR PROTECTING AGAINST HARMFUL CHEMICAL AGENTS; CHEMICAL MATERIALS FOR USE IN BREATHING APPARATUS
    • A62D2101/00—Harmful chemical substances made harmless, or less harmful, by effecting chemical change
    • A62D2101/20—Organic substances
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F2101/00—Nature of the contaminant
    • C02F2101/30—Organic compounds
    • C02F2101/32—Hydrocarbons, e.g. oil
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales

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Abstract

The present invention relates to microorganism fields, in particular to a kind of vinyon efficient degrading bacteria and its separating screening method and the application in terms of vinyon biodegrade.The bacterial strain is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number are as follows: CGMCCNo:15724；The preservation time are as follows: on May 4th, 2018.The bacterial strain has good environmental suitability, has to the biodegrade aspect of vinyon, especially sealed polyethylene plastic and is widely applied very much, provides new resources for the biodegrade of vinyon, have a good application prospect.

Description

A kind of vinyon efficient degrading bacteria and its separating screening method and application
Technical field
The present invention relates to microorganism fields, in particular to a kind of vinyon efficient degrading bacteria and its bolter Choosing method and the application in terms of vinyon biodegrade.
Background technique
Plastics are to process the organic compound synthesized by petroleum chemicals, due to its excellent chemical stability and mechanical performance And its features such as durability and economy, it is used widely in life.Plastics are hardly decomposed after disposition, in environment In persistent accumulation cause serious ecological threat, " white pollution " has become global problem.Polyethylene (Polyethylene, PE) is one of yield and the highest synthetic polymer of dosage in plastic products, and global yield is every year about It is 80,000,000 tons.PE basic structure is-[CH2-CH2]n, since its relative molecular mass is larger, hydrophobicity is strong, especially highly The enzyme of stable C-C skeleton and shortage Direct Pyrolysis C-C chain, biological degradability is poor in the natural environment, easily accumulates in the environment.
It is mostly at present landfill, burning, secondary operation etc. to the disposal options of waste plastic, these processing modes are to environment Pollution it is larger, processing cost is high, and easily causes secondary pollution.Therefore, other than researching and developing new environmental-friendly plastic products, The processing technique of the plastic refuses safe green such as polyethylene disposition is urgently researched and developed.Nowadays for plastics such as polyethylene The research of waste biological treating method is less, and efficient bacterium resource is few, and governance efficiency is low, limits its further extension and answers With.
Summary of the invention
It is an object of that present invention to provide a kind of vinyon efficient degrading bacteria and its separating screening method and in polyethylene Application in terms of Study of Biodegradation of Common.
To achieve the above object, the invention adopts a technical scheme as:
One plant of vinyon efficient degrading bacterial strain, vinyon efficient degrading bacterial strainRaoultella ornithinolyticaMP-1 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, address Are as follows: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Institute of Microorganism, Academia Sinica, deposit number CGMCC No: 15724；The preservation time are as follows: on May 4th, 2018.
A kind of separating screening method of vinyon efficient degrading bacterial strain turns by sample to be separated by enrichment culture It is connected to using polyethylene as enrichment isolation, separation in the culture medium of sole carbon source, and then obtaining one plant can be carbon using polyethylene The degradation bacteria MP-1 in source and the energy.
The sample to be separated is environment plastic refuse.
The polyethylene is the nutrient media components of sole carbon source are as follows: K2HPO4·3H2O 0.7 g, KH2PO40.7 g, NH4NO31.0 g, NaCl 0.5 g, MgSO4·7H2O 0.5 g, FeSO4·7H2O 0.002 g, ZnSO4·7H2O 0.002 G, MnSO4·H2O 0.001 g；Mentioned reagent is dissolved in 1 L deionized water, adjusting pH to 7.0 with 1 mol/L NaOH is For fluid nutrient medium；Agar by addition 1.5% in mentioned reagent is solid medium.
A kind of application of vinyon efficient degrading bacterial strain, the polyethylene degradation bacterial strain MP-1 are poly- in biodegrade Application in ethylene.
The application of polyethylene degradation bacterial strain MP-1 Biodegradable polyethylene film in water body.
The method for separation screening vinyon degradation bacteria that the present invention relates to a kind of from environment plastic refuse, passes through richness Collection culture, is forwarded to using polyethylene as enrichment isolation in the culture medium of sole carbon source, repeatedly scribing line separation, and being separated to one plant can It is the degradation bacteria MP-1 of carbon source and the energy, the colonial morphology Expressive Features of the bacterial strain are as follows: bacterium colony is faint yellow, shape using polyethylene For circle, the smooth wet, neat in edge in surface is clear-cut.The aobvious red of Gram's staining, is negative.To bacterial strain 16S rRNA Gene order has carried out amplification sequencing, sequence existing in resulting sequence and ncbi database is carried out BLAST analysis, as a result It has been shown that, MP-1 withRaoultella ornithinolytica(AJ251467) similitude is 99%, and the phase on evolutionary distance To relatively close, in conjunction with the physiological and biochemical property of bacterial strain, tentatively by MP-1 be accredited as solution ornithine Raoul bacterium (Raoultella ornithinolyticaMP-1).
Vinyon degradation bacteria provided by the present application (Raoultella ornithinolytica), the entitled MP- of bacterial strain 1, it is isolated from the plastic refuse surface of harbour floating, is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms Center, deposit number CGMCC No:15724；The preservation time are as follows: be detected as survival strains and preservation on May 4th, 2018.
Advantage for present invention:
The strain of polyethylene degradation can be provided using the separating screening method and then acquisition of plastic degradation bacterium of the invention Source, the present invention, which obtains bacterial strain, has good effect for vinyon biodegrade aspect.The method of the present invention is environmentally protective, At low cost, easy to operate, obtained strains have good degradation characteristic, provide for the biological prosthetic of polyethylene waste in environment New resources and thinking, have a extensive future.
Detailed description of the invention
Fig. 1 is polyethylene degradation bacterium provided by the inventionRaoultella ornithinolyticaThe bacterium colony shape of MP-1 State characteristic pattern.
Fig. 2 is the growth curve chart of polyethylene degradation bacterium MP-1 provided by the invention.
Fig. 3 is the phylogenetic tree of polyethylene degradation bacterium MP-1 provided by the invention.
PE film is in optical microscopy (a control after Fig. 4 is polyethylene degradation bacterium provided by the invention degradation 30 days；B connects bacterium MP-1) and scanning electron microscope (c control；D meets bacterium MP-1) shape characteristic figure.
Fig. 5 is the water contact angle variation diagram of PE film before and after polyethylene degradation bacterium provided by the invention is degraded.
Fig. 6 is PE film surface functional group variation diagram before and after polyethylene degradation bacterium provided by the invention is degraded.
Specific embodiment
It, below will be to specific in order to illustrate more clearly of the specific embodiment of the invention or technical solution in the prior art Embodiment or attached drawing needed to be used in the description of the prior art be briefly described, it should be apparent that, it is described below Attached drawing is some embodiments of the present invention, for those of ordinary skill in the art, before not making the creative labor It puts, is also possible to obtain other drawings based on these drawings.
Polythene PE efficient degrading bacteria of the present inventionRaoultella ornithinolyticaIt is micro- to be preserved in China by MP-1 Biological inoculum preservation administration committee common micro-organisms center, address are as follows: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 China Institute of microbiology, the academy of sciences, deposit number CGMCC No:15724；The preservation time are as follows: on May 4th, 2018.
The bacterial strain to plastic refuse have good degradation effect, especially for polyethylene film degradation effect compared with Good, this method is environmentally protective, at low cost, easy to operate.By using this biodegradable method, polyethylene after processing 30 days Film surface has apparent bioerosion hole, and diaphragm hydrophily is obviously improved, and surface fracture is serious, weight-loss ratio after processing 60 days Up to 4.37 ± 0.66%, new resources and thinking are provided for the biological prosthetic of polyethylene waste, are had a extensive future.
The MP-1 bacterial strain of institute's preservation of the present invention has very strong polyethylene degradation ability, is starting strain suitable using the bacterial strain It spends in range and mutates, obtained mutant strain still has very strong polyethylene degradation ability.
And during practical application, it is contemplated that it may need the reasons such as transport, the bacterium that above-mentioned screening is obtained Strain, and mutation obtain bacterial strain individually or any combination, according to fermented and cultured mode expand culture gained pure culture Or the form of mixed culture (using pure culture or mixed culture as microbial bacterial agent) can be carried out further to poly- Ethylene PE degrades, and is suitable for large-scale practical application, to expand its application range.
Embodiment 1
The separation screening of polyethylene degradation bacterium and identification
(1) separation screening of polyethylene degradation bacterium
Separation screening is carried out as sample to be separated using the plastic refuse floated from harbour；
It will be cut into small pieces, be placed in sterile conical flask after collected plastic refuse aseptic water washing, is added 0.9% physiological saline and bead is prepared into bacterium 28 DEG C in constant-temperature shaking incubator, 180 r/min shaken cultivation, 2 h Suspension.By bacteria suspension with 2% inoculum concentration (v/v) be forwarded in carbon-free source mineral salt culture medium, the polyethylene of ultraviolet sterilization is added Powder (2 g/L), 28 DEG C, 180 r/min shaken cultivation, 15 d, 3 groups of setting are parallel.The OD of period sampling measuring system600Value, Cultivating system continues to transfer after becoming cloudy, and repeats culture 3 times.Selecting the rapid cultivating system of growth, gradient dilution is prepared not step by step Same dilution (10-3、10-4、10-5、10-6) bacteria suspension, with dilution spread flat band method isolated strains, and repeatedly scribing line purifying point From the bacterial strain arrived.After the single colonie bacterial strain being separated to is saved, verifying is to polyethylene on carbon-free source mineral salt solid medium Utilization ability.No carbon source solid plate surface is coated with sterilized polyethylene powders, and the bacterial strain of acquisition is crossed on surface and is trained It supports, selects the rapid single bacterium of growth and fall within culture preservation on LB plate.
The culture medium of separation screening is carbon-free source mineral salt culture medium, component are as follows: K2HPO4·3H2O 0.7 g, KH2PO4 0.7 g, NH4NO3 1.0 g, NaCl 0.5 g, MgSO4·7H2O 0.5 g, FeSO4·7H2O 0.002 g, ZnSO4·7H2O 0.002 g, MnSO4H2O 0.001 g.Mentioned reagent is dissolved in 1 L deionized water, adjusts pH with 1 mol/L NaOH To 7.0.1.5% agar is added in solid medium.Addition polyethylene is sole carbon source.
By above-mentioned separation and screening operation, separating for several times purifying obtains one plant of rapid polyethylene degradation bacterium of growth MP-1.Bacterium colony is in faint yellow on LB plate for the degradation bacteria, and shape is circle, and surface is smooth wet, neat in edge (Fig. 1).
(2) physiological characteristic and molecular biology identification of bacterial strain
Gram's staining is carried out to the above-mentioned thallus of culture to logarithmic phase.
The DNA that the single bacterial strain that above-mentioned separation obtains is extracted with kit, using bacterial universal primers 27F, 1492R conduct Amplimer carries out PCR amplification.
16S rDNA aligning primer 27F:5'- AGA GTT TGA TCC TGG CTC AG- 3', 1492R:5'- GGT TAC CTT GTT ACG ACT T- 3'。
PCR amplification system (50 μ L): template DNA 5 μ L, Mix (2 ×) 25 μ L, primer 2 7F 2 μ L, primer 1492R 2 μ L, ddH2O 16 μL.Amplification program: 95 DEG C of initial denaturations 5 min, 95 DEG C of 1 min of denaturation；58 DEG C of 1 min of annealing；72 DEG C are prolonged 2 min are stretched, are recycled 30 times；10 min of last 72 DEG C of extensions, 4 DEG C of preservations.Electrophoresis detection is carried out to amplified production, the commission English Weihe River is prompt The sequencing of base (Beijing) biotech firm.Sequence existing in resulting sequence and ncbi database is subjected to BLAST analysis, and is chosen Bacterial strain similar in homology, using 6.0 software building phylogenetic tree of MEGA (referring to Fig. 3).
Under optical microscopy, bacterial strain is negative through the aobvious red of Gram's staining.Growth curve of the MP-1 in LB culture medium As shown in Fig. 2, MP-1 is grown rapidly in LB culture medium, logarithmic growth phase is initially entered after 2 h.16S rDNA sequence alignment knot Fruit shows, MP-1 withRaoultella ornithinolytica(AJ251467) similitude is 99%, and on evolutionary distance MP-1 is tentatively accredited as solution ornithine Raoul bacterium in conjunction with the physiological and biochemical property of bacterial strain by relative close (Fig. 3) (Raoultella ornithinolyticaMP-1).
Embodiment 2
Polyethylene degradation bacterium follows the steps below the degradation experiment of polyethylene film:
For application potential of the assessment polyethylene degradation bacterium in real life, choose polyethylene film common in life into Row degradation experiment.
MP-1 is seeded in LB culture medium by the inoculum concentration of 2-5% and is cultivated to logarithmic growth phase, with 0.01 mol/L phosphoric acid Salt buffer solution washs the culture medium on 3 times removal surfaces, is resuspended again with carbon-free source mineral salt culture medium later, then adjusts and is resuspended Bacterium solution OD600Value is 1.0.Polyethylene film pieces are cut into 2 × 2 cm2Size, after weighing with 75% ethyl alcohol sterilize 2 h, after taking-up The ethyl alcohol on surface is vapored away with germ-free air flow in super-clean bench.The carbon-free source mineral salt culture medium of 90 mL is added in conical flask, is added The polyethylene film pieces (2 g/L) of surface sterilizing, with 10%(v/v) inoculum concentration be inoculated with above-mentioned MP-1 resuspended bacterium solution.Not connect at bacterium Reason is control group, and 3 Duplicate Samples are arranged in every kind of processing.Conical flask is placed in 28 DEG C, 180 r/ in isothermal vibration incubator Min culture, pattern variation, functional group's variation, mass loss and the hydrophobicity variation on regular sampling analysis polyethylene film pieces surface (referring to fig. 4).
Polyethylene film pieces surface microscopic topographic changes according to following flow processing: with the culture of aseptic water washing excess surface Base fixes 2 h with 2.5% glutaraldehyde, then successively with 30%, 50%, 70%, 90%, 100% Gradient elution using ethanol, every time 15 min, then It is replaced 2 times with the tert-butyl alcohol, 30 min every time.The sample drying that will be handled well is fixed after metal spraying in scanning electron microscope Its microscopic appearance feature is observed under (Hitachi S-4800 FE-SEM, Hitachi).
The mass change of polyethylene film pieces is according to following flow processings: vibrating washing polyethylene film pieces 4 with 2% SDS solution H, 15 min of ultrasound remove the biomembrane on surface, then with sterile water washing diaphragm 3 times, and the polyethylene film pieces after washing are placed It weighs after dry 48 h in drier.
The mass attenuation rate (%) of polyethylene=(quality after polyethylene initial mass-degradation)/initial mass × 100%
The measurement of polyethylene film pieces surface functional group is measured using Fourier infrared spectrograph, the diaphragm cleaned up It is measured after natural drying.Scanning wavelength range 600-4000 cm-1, 4 cm of resolution ratio-1, scanning times 32 times.
With contact angle measurement measurement contact angle characterization film surface hydrophobicity variation (referring to Fig. 5).
From fig. 4, it can be seen that by the culture of 30 d, is observed under scanning electron microscope and connect the diaphragm of bacterium processing compared with the control group There is disintegration in rough surface, edge, and apparent microbial attack hole occurs in surface.And it as seen from Figure 5, is surveyed through contact angle Amount instrument the results show that connect bacterium processing polyethylene film pieces water contact angle be 74.4 ± 0.3 °, far below control treatment 93.5 ± 2.4 °, show that connecing bacterium processing significantly reduces the hydrophobicity of PE film, it is easier to be adhered to by microorganism and corrode.Simultaneously by Fig. 6 FTIR is the results show that new characteristic peak occurs in PE film after degradation, in 1716 cm-1 Carbonyl peak is measured.Carbonyl [- C=O- Key] (1720cm-1Left and right) appearance be that biodegradable basic sign occurs for PE, show that biodegrade has occurred in polyethylene.And After continue according to after above-mentioned training method culture to 60 d, the diaphragm quality of inoculation MP-1 processing reduces 4.37 ± 0.66%, and Control treatment does not change, shows to degrade polyethylene using MP-1.
Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention., rather than its limitations；To the greatest extent Present invention has been described in detail with reference to the aforementioned embodiments for pipe, but those skilled in the art should understand that: its It is still possible to modify the technical solutions described in the foregoing embodiments, or to some or all of the technical features It is equivalently replaced；And these are modified or replaceed, various embodiments of the present invention skill that it does not separate the essence of the corresponding technical solution The range of art scheme.

Claims (3)

1. one plant of vinyon efficient degrading bacterial strain, which is characterized in that vinyon efficient degrading bacterial strainRaoultellaornithinolyticaMP-1 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms Center, deposit number CGMCC No:15724；The preservation time are as follows: on May 4th, 2018.
2. a kind of application of vinyon efficient degrading bacterial strain described in claim 1, it is characterised in that: the polyethylene Application of the degradation bacteria strains MP-1 in Biodegradable polyethylene.
3. the application of vinyon efficient degrading bacterial strain as described in claim 2, it is characterised in that: the polyethylene drop Solve the application of bacterial strain MP-1 Biodegradable polyethylene film in water body.
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CN115322941B (en) 2022-10-14 2022-12-27 海南热带海洋学院 Rose color bacterium, application and culture method thereof, and method for degrading plastics
CN115322942B (en) * 2022-10-14 2022-12-09 海南热带海洋学院 Hay bacillus, application and culture method thereof and method for degrading plastics
CN115960759B (en) * 2022-10-14 2024-05-14 西南大学 Polystyrene plastic and polyethylene plastic degrading bacterium, screening method and application thereof
CN115354008B (en) * 2022-10-14 2022-12-23 海南热带海洋学院 Paecilomyces periplaneta, application and culture method thereof, and method for degrading plastics
CN115960782B (en) * 2022-12-22 2023-09-26 中国科学院南海海洋研究所 Marine polyethylene plastic degrading bacterium and application thereof
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