CN117821314A - Polyethylene plastic degrading bacterium screening method and application - Google Patents

Polyethylene plastic degrading bacterium screening method and application Download PDF

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Publication number
CN117821314A
CN117821314A CN202311836478.3A CN202311836478A CN117821314A CN 117821314 A CN117821314 A CN 117821314A CN 202311836478 A CN202311836478 A CN 202311836478A CN 117821314 A CN117821314 A CN 117821314A
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polyethylene
strain
polyethylene plastic
medium
plastic
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夏伟
吴敬
姚从禹
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Jiangnan University
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Jiangnan University
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Abstract

The invention discloses a screening method and application of polyethylene plastic degrading bacteria, and belongs to the field of microorganisms. The invention provides a screening method of polyethylene plastic degrading bacteria, and 2 strains with polyethylene degrading capability are successfully screened and obtained. And the separation and screening method and the application thereof in the aspect of biodegradation of polyethylene plastics. The obtained strain has good environmental adaptability, has wide application in the aspect of biodegradation of polyethylene plastics, in particular to polyethylene plastic films, provides new resources for the biodegradation of polyethylene plastics, and has good application prospect.

Description

Polyethylene plastic degrading bacterium screening method and application
Technical Field
The invention relates to a polyethylene plastic degrading bacterium screening method and application thereof, belonging to the field of microorganisms.
Background
Polyethylene in petroleum-based plastics is widely used in various fields of foods, construction, agriculture, etc. by virtue of its excellent durability, and the global yield increases exponentially. It is difficult to degrade effectively in natural environments because of its strong chemical inertness. The "white pollution" caused by plastics has become a "big and difficult" problem for the global environment. Most of the plastic waste is released into the environment from land to water circle everywhere due to the inefficient recovery of waste plastic.
At present, non-biological treatment methods such as landfill incineration and the like are generally adopted at home and abroad, so that resource waste and secondary pollution are easy to cause, and the biodegradable plastic method has strong potential for ecological sustainable development and environmental protection. Therefore, in addition to the development of new environmentally friendly plastic products, there is a need for research and development of a treatment technology for safely and environmentally friendly disposal of plastic wastes such as polyethylene. At present, the biological treatment method for polyethylene and other plastic wastes has less research, few effective strain resources and low treatment efficiency, and further expansion application of the method is limited.
Disclosure of Invention
The invention aims to provide a polyethylene plastic high-efficiency degradation bacterium, a separation and screening method thereof and application thereof in the aspect of biodegradation of polyethylene plastic.
The invention provides a strain with polyethylene degradation capability, which is (a) or (b):
(a) Ochrobactrum_inter (Ochrobactrum) D2-6-1 was deposited at China Center for Type Culture Collection (CCTCC) No: m20231435;
(b) Pseudomonas (Pseudomonas sp.) D4-2-1 was deposited with the China center for type culture Collection, with a accession number CCTCC NO: m20231434.
The invention also provides a microbial preparation containing the strain.
In one embodiment, the microbial preparation contains living cells of the Ochrobactrum intermedium D2-6-1 and/or the Pseudomonas aeruginosa D4-2-1.
The invention also provides a method for producing oxidase by using the strain, which is to culture the strain in a culture medium under an aerobic environment at 25-35 ℃.
In one embodiment, the medium in which the strain is cultivated contains a carbon source, a nitrogen source and an inorganic salt; the plastic may be provided as the sole carbon source or with other carbon sources; the nitrogen source may be an organic nitrogen source or an inorganic nitrogen source, including but not limited to peptone, meat extract, yeast extract, corn steep liquor, sulfate, ammonium salt, etc.; the medium may further contain an inorganic salt composed of cations such as sodium ion, potassium ion, calcium ion, magnesium ion, and anions such as sulfate ion, chloride ion, phosphate ion, and the like.
In one embodiment, the medium further comprises trace elements such as vitamins, nucleic acids, and the like.
In one embodiment, the method is to culture the strain in NB medium for a period of time and then transfer to medium with polyethylene as the sole carbon source, at 30℃at 180-200 rpm for 10-20 d.
The invention also provides a separation and screening method of the polyethylene plastic degradation strain, which comprises the steps of transferring a sample to be separated into a culture medium taking polyethylene as a unique carbon source for culture for a period of time through domestication and enrichment culture, and collecting the strain with polyethylene degradation capability.
In one embodiment, the acclimation and enrichment culture is performed in mineral salt medium without a carbon source.
In one embodiment, the mineral salt medium without carbon source contains: k (K) 2 HPO 4 ·3H 2 O 0.5-1.0g/L，KH 2 PO 4 0.5-1.0g/L，NH 4 NO 3 1.0g/L，NaCl 0.5g/L，MgSO 4 ·7H2O 0.3-0.7g/L，FeSO4·7H 2 O 0.002g/L，ZnSO 4 ·7H 2 O 0.002g/L，MnSO 4 ·H 2 O 0.001g/L，CuSO 4 0.001g/L。
The invention also provides application of the polyethylene plastic degradation strain in the aspects of polyethylene plastic product degradation, polyethylene plastic biological circulation, polyethylene plastic upgrading and reconstruction, polyethylene plastic surface modification and the like.
In one embodiment, the use is to depolymerize the polyethylene.
In one embodiment, the application is to contact the polyethylene plastic degrading strain with polyethylene plastic for a period of time to depolymerize the polyethylene.
In one embodiment, the polyethylene plastic article is subjected to ultraviolet radiation treatment.
In one embodiment, the ultraviolet irradiation treatment is to subject the polyethylene plastic article to ultraviolet irradiation at 275 to 200nm at 60 to 80 ℃ for at least 1d, or at least 2d, or at least 3d.
In one embodiment, the ultraviolet irradiation treatment is to subject the polyethylene plastic article to ultraviolet irradiation at 275-200nm for 3d at 70 ℃.
In one embodiment, the use is to culture the polyethylene plastic degrading strain at 25 to 35 ℃ for at least 24 hours, or 36 hours, or 48 hours, or 60 hours, or 72 hours in an environment containing the polyethylene plastic article.
In one embodiment, the use is to culture the polyethylene plastic-degrading strain at 30 ℃ for at least 30d in an environment comprising the polyethylene plastic article.
The beneficial effects are that:
1. the invention provides an intermediate pallidum D2-6-1 and pseudomonas D4-2-1 with polyethylene plastic degradation capability, which has the following new characteristics:
(1) The polyethylene degradation and utilization capability is provided, the polyethylene can grow in a culture medium taking polyethylene as a sole carbon source, and polyethylene materials can be degraded in a solution environment;
(2) Capable of exogenously secreting oxidase, hydrolyzable ABTS or DMP;
(3) Has the coding genes of oxidase such as laccase, alkane hydroxylase and the like.
2. The invention also provides a method for degrading polyethylene by applying the strain, which is environment-friendly, low in cost and convenient to operate, provides new resources and ideas for bioremediation of polyethylene wastes in the environment, and has wide application prospect.
Preservation of biological materials
Ochrobactrum_inter D2-6-1, which is classified and named as Ochrobactrum_inter D2-6-1, is preserved in China center for type culture collection, and has a preservation number of CCTCC NO: m20231435; the preservation time is 2023, 8 and 11 days, and the preservation address is China, wuhan, university of Wuhan.
Pseudomonas (Pseudomonas sp.) D4-2-1, classified and named as Pseudomonas sp.D4-2-1, is preserved in China center for type culture Collection, and has a preservation number of CCTCC NO: m20231434; the preservation time is 2023, 8 and 11 days, and the preservation address is China, wuhan, university of Wuhan.
Drawings
FIG. 1 shows growth of polyethylene degrading bacteria obtained by screening and separation in a sole carbon source culture medium of UVPE; a: a control group; b: ochrobactrum_inter D2-6-1; c: pseudooxantahomonas_sp D4-2-1.
FIG. 2 shows the growth curve of polyethylene degrading bacteria with UVPE as the sole carbon source.
FIG. 3 is an SEM image of a UVPE membrane after treatment with polyethylene degrading bacteria; control: a, D; ochrobactrum_inter D2-6-1: b, E; pseudooxantahomonas_sp D4-2-1.
Fig. 4 is an infrared image of a UVPE patch after treatment with polyethylene degrading bacteria.
FIG. 5 shows the possible degradation products detected by GCMS headspace.
FIG. 6 is a SDS-PAGE protein gel of extracellular secreted protein after UVPE treatment with degrading bacteria.
FIG. 7 shows the change in extracellular secretion oxidase enzyme activity of two polyethylene degrading bacteria during incubation of UVPE; a, ABTS is taken as a substrate, B, DMP is taken as a substrate.
Detailed Description
The LDPE film referred to in the examples below was purchased from China Petroleum and Natural gas Co., ltd and had a thickness of 20. Mu.m. 2, 2-nitrogen-bis (3-ethylbenzothiazole-6-sulfonic Acid) (ABTS) and 2,6,2 methoxyphenol (DMP) were purchased from Sigma-Aldrich (china). Reagents such as ethanol and ethyl acetate are purchased from national medicine reagents. All chemicals reached reagent grade purity.
The following examples relate to the following media:
NB medium: tryptone 10.00g.L -1 ，NaCl 5.00g.L -1 Beef extract 3.00g.L -1 Agar powder 20.00g.L is added into the solid culture medium -1 。
Carbon source free mineral salt medium: k (K) 2 HPO 4 ·3H 2 O 0.5-1.0g.L -1 ，KH 2 PO 4 0.5-1.0g.L -1 ，NH 4 NO 3 1.0g.L -1 ，NaCl 0.5g.L -1 ，MgSO 4 ·7H2O 0.3-0.7g.L -1 ，FeSO 4 ·7H 2 O 0.002g.L -1 ，ZnSO 4 ·7H 2 O 0.002g.L-1，MnSO 4 ·H 2 O 0.001g.L-1，CuSO 4 0.001g.L -1 Agar powder 20.00g.L is added into the solid culture medium -1 。
The detection method involved in the following examples is as follows:
the detection method of oxidase enzyme activity comprises the following steps:
1 enzyme activity unit (U) is defined as the amount of enzyme required to oxidize 1. Mu. Mol of substrate in 1 min.
Enzyme activity determination with ABTS as substrate, 10mM ABTS solution (4 μl) and citric acid-hydrochloric acid buffer (186 μl; pH 5.0) are added into 96-well ELISA plate, and the ELISA apparatus is set to 30deg.C for system preheating; 10. Mu.L of the enzyme solution was added, the degree of change in OD at 420nm was measured, and the enzyme activity was calculated.
Enzyme activity assay with DMP as substrate: 20mM DMP solution (4. Mu.L) and citric acid-hydrochloric acid buffer (186. Mu.L; pH 5.0) were added to a 96-well microplate, and the microplate reader was set to 30℃for systematic pre-heating; 10. Mu.L of the enzyme solution was added, the degree of change in OD at 470nm was measured, and the enzyme activity was calculated.
The detection method of the microstructure of the surface of the Polyethylene (PE) plastic film comprises the following steps:
repeatedly cleaning a polyethylene film (UVPE) treated by degrading bacteria with deionized water for 3-4 times; the washed film is sequentially washed twice with 2% SDS (v/v), deionized water and 50% ethanol respectively by ultrasonic method to remove impurities such as protein on the surface of the film for 30min each time. After the rinsing is finished, the membrane is wrapped in weighing paper and is placed in a baking oven at 50 ℃ for drying. The surface morphology and chemical structure changes of the untreated ultraviolet pretreated polyethylene film (UVPE) and the ultraviolet pretreated polyethylene film (UVPE) after treatment with a laccase mediator system were detected using a Scanning Electron Microscope (SEM), fourier transform infrared spectroscopy (FTIR) and Differential Scanning Calorimeter (DSC) as a control.
Detection of degradation products of UVPE:
the type and proportion of small molecular substances in the reaction solution were detected using a gas-high throughput time-of-flight mass spectrometer (TSQ 8000, thermo; U.S.). The gas phase detection conditions are as follows: DB-5MS chromatographic column, sample injection speed 1.0ml/min, initial temperature 40 ℃, heating up to 250 ℃ at a heating rate of 10 ℃/min and maintaining for 4min. The mass spectrum detection conditions are as follows: molecular weight detection range is 33-450, ion source temperature is 70 ℃, and electron energy is 70.0.
Solid phase microextraction method: the extraction head is adopted: DVB (Sigma-Aldrich) was inserted into the headspace bottle for target adsorption, after 7min of adsorption, the needle was inserted into the GC inlet for injection, and the process was performed at a constant helium flow (1.0 ml/min).
Example 1: separation screening and identification of polyethylene
Taking compost plastic waste from a refuse landfill as a sample to be separated for separation and screening;
the collected plastic waste is washed by sterile water and cut into small blocks, the small blocks are placed in a sterile conical flask, 0.9% physiological saline and glass beads are added, and the small blocks are subjected to shaking culture for 2 hours at 30 ℃ and 150r/min in a constant-temperature shaking incubator, so that bacterial suspension is prepared.
Domestication culture: the bacterial suspension was transferred to a carbon-free mineral salt medium at an inoculum size (v/v) of 2%, liquid paraffin (2 g/L) was added thereto, and the culture was performed at 30℃and 150r/min with shaking for 7d.
Enrichment culture: 5mL of the domesticated solution was aspirated and added to 15mL of a medium containing a unique carbon source mineral salt of ultraviolet irradiated polyethylene powder. The pretreated powder is washed 3-5 times with deionized water, washed and soaked with 1% SDS solution, then washed 2 times with 75% ethanol, and finally dried in a 40 ℃ oven. The powder is directly mixed with sterilized mineral salt culture medium without high temperature sterilization, and shaking. Culturing at 30deg.C and 150r/min for 7d (liquid turbidity or OD value increase is observed, and the next step can be performed). Then 2mL of the enrichment was aspirated and added to a new 18mL of mineral salt medium of the sole carbon source containing untreated polyethylene powder, and incubated under the same conditions until the turbidity of the liquid increased.
And (3) separating and screening: selecting a rapidly growing culture system to prepare different dilutions (10) -3 、10 -4 、10 -5 、10 -6 ) The strain is isolated by a dilution-spread plate method, and the isolated strain is purified by streaking a plurality of times. After the isolated single colony strains were stored, the polyethylene availability was verified on carbon source free mineral salt solid and liquid media (FIG. 1). Genome analysis is carried out on the strains with polyethylene utilization capability, wherein the 16S rDNA sequence of the Ochrobactrum_inter medium D2-6-1 is shown as SEQ ID NO. 1; the 16S rDNA sequence of pseudooxantahomonas_sp D4-2-1 is shown as SEQ ID NO. 2.
Genome sequencing and gene prediction: the Illumina HiSeq and PacBio sequence system instruments and related software used for whole genome sequencing of polyethylene degrading strains are provided by Shanghai Biotechnology Co. Based on the predicted coding sequences, the BLAST program was used to align the sequences with the database, respectively. And carrying out functional annotation on the coding genes by using databases such as NR, KEGG, COG, swissprot, CDD, GO and the like, and selecting an optimal comparison result as an annotation result of the coding genes.
Example 2: culture of polyethylene degrading bacteria and preparation of microbial inoculum
Inoculating the strain obtained in the screening of example 1 into NB medium at an inoculum size of 2-5%The medium of the surface is then resuspended in a mineral salt medium without a carbon source. Adding 90mL of carbon-free mineral salt culture medium into a conical flask, adding surface sterilized polyethylene membrane (2 g/L), inoculating seed solution into the carbon-free culture medium (inoculated strain OD) 600 0.09), and culturing at 30deg.C and 180r/min in a constant temperature shake incubator.
Example 3: biodegradation of ultraviolet pretreated polyethylene film by polyethylene degrading bacteria
In order to evaluate the application potential of the polyethylene degrading bacteria in actual life, a common polyethylene preservative film in life is selected for degradation experiments. The PE plastic film is flatly paved on a stainless steel bracket, and is put into an ultraviolet aging box (UVC, 275-200 nm) to set the aging temperature to be 70 ℃. Cutting polyethylene film after irradiation treatment for 3 days into 2×2cm 2 The membrane is soaked in 75% ethanol for sterilization for 2 hours by using sterilized forceps, and the ethanol on the surface is volatilized by using sterile air flow in an ultra-clean bench after being taken out and used as a substrate for biodegradation experiments.
The strain obtained by screening in example 1 was inoculated into NB medium in an inoculum size of 2-5%o to the logarithmic phase, washed 3 times with physiological saline to remove the surface medium, and then resuspended in a mineral salt medium without carbon source. Adding 90mL of carbon-free mineral salt culture medium into a conical flask, adding surface sterilized polyethylene membrane (2 g/L), inoculating seed solution into the carbon-free culture medium (inoculated strain OD) 600 0.09). The control group was a sterile treatment, and 3 replicates were set for each treatment. The conical flask was placed in a constant temperature shaking incubator at 30℃and 180 r/min.
During the culture, samples are taken and measured on a super clean bench at regular intervals for OD of each strain 600 Growth conditions and oxidase enzyme activity in the culture medium. The strain growth condition is shown in figure 2, the oxidase enzyme activity change is shown in figure 7, after 60 days of degradation experiment, fermentation broth of microorganism growth is collected by using a nylon net for filtration, and the semi-volatile and volatile components in the components are determined by a GCMS headspace method. The polyethylene film was taken out from the reaction-terminated polyethylene film with clean tweezers, and the film was ultrasonically cleaned by immersing it in 2% SDS, deionized water and 50% ethanol, respectively. Washed wellAfter the film was oven dried, SEM, FTIR and DSC measurements were performed.
The Ochrobactrum_inter D2-6-1,Pseudoxanthomonas_sp D4-2-1 can grow in mineral salt culture medium with only carbon source of UVPE, which shows that both strains can grow and metabolize with UVPE as nutrition component.
As shown in FIG. 3, the membrane after microbial degradation has the roughness and homolysis phenomenon of different layers. The experimental group showed that Ochrobactrum_inter D2-6-1,Pseudoxanthomonas_sp D4-2-1 had an effective biodegradation effect on polyethylene films as compared to the control group.
As shown in FIG. 4, the polyethylene film treated with Ochroctrum_inter D2-6-1,Pseudoxanthomonas_sp D4-2-1 was 1000-1200cm -1 ，1500-1700cm -1 At 3300cm -1 The new peak which is not found in the control group appears in the nearby wave number range, and the peak in the three wave number ranges corresponds to the peak position of each oxygen-containing tube group such as hydroxyl, carbonyl and the like, which also shows the deep oxidation capability of four polyethylene degrading bacteria to the polyethylene membrane and is corresponding to the oxidase enzyme activity detected in the culture process.
The crystallinity results show a slight increase in crystallinity of the membrane after the microbial treatment, which also indicates the preferential use of the polymer vulnerable portion by the microorganisms. As shown in FIG. 5 and Table 1, with the degradation of polyethylene film by Ochrobacterium_inter D2-6-1,Pseudoxanthomonas_sp D4-2-1, degradation and metabolites such as alkane, ketone, alcohol, ester, acid and the like can be detected from the fermentation broth, further illustrating the degradation ability of two strains of polyethylene degrading bacteria to polyethylene film.
In addition, it was found by protein electrophoresis detection of the degraded broth (FIG. 6) that Ochrobactrum_inter D2-6-1,Pseudoxanthomonas_sp D4-2-1 secreted a large number of extracellular proteins during degradation of UVPE. The process of biodegradation of polyethylene is initially accompanied by a progressive increase in the extent of oxidation of its carbon chains, which in turn effects depolymerization of the carbon chains to a certain extent. And small molecular substances depolymerized from the carbon chains are absorbed into cells for growth and utilization through a transport system of the degrading bacteria pair.
The enzyme activity of extracellular oxidase was detected using universal substrates ABTS and DMP for both oxidases, and as can be seen in fig. 7, oxidase was present during the growth process. As can be seen from the whole genome sequencing of Ochrobacterium_inter D2-6-1,Pseudoxanthomonas_sp D4-2-1 and from the blast comparison with the data, both strains have a certain number of oxidases including laccase, alkane hydroxylases, and enzymes involved in transmembrane transporters and intracellular metabolism of degradation products, etc. (Table 2, 3). These functional proteins all demonstrate the ability of Ochrobactrum_Intermedia D2-6-1,Pseudoxanthomonas_sp D4-2-1 to biodegrade polyethylene at the gene level.
TABLE 1 UVPE crystallinity after microbial degradation
Control D2-6-1 D4-2-1
Crystallinity (%) 17.5 19.5 18.9
Table 2Ochrobactrum_intermedium D2-6-1 functional proteins related to polyethylene degradation in genome
Table 3Pseudoxanthomonas_sp D4-2-1 functional proteins related to polyethylene degradation in genome
While the invention has been described with reference to the preferred embodiments, it is not limited thereto, and various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

Claims (12)

1. A strain having polyethylene degrading ability, characterized by being (a) or (b):
(a) Ochrobactrum_inter (Ochrobactrum) D2-6-1 was deposited at China Center for Type Culture Collection (CCTCC) No: m20231435;
(b) Pseudomonas (Pseudomonas sp.) D4-2-1 was deposited with the China center for type culture Collection, with a accession number CCTCC NO: m20231434.
2. A microbial preparation comprising the strain of claim 1.
3. The microbial preparation according to claim 2, wherein the microbial preparation contains living cells of the pallidum D2-6-1 and/or the pseudomonas D4-2-1.
4. A method for producing oxidase by using the strain according to claim 1, wherein the strain is cultured in a medium at 25 to 35℃in an aerobic environment.
5. The method according to claim 4, wherein the strain is cultured in NB medium for a period of time and then transferred to a medium with polyethylene as the sole carbon source, and cultured at 180-200 rpm at 30℃for 10-20 d.
6. A method for separating and screening polyethylene plastic degradation bacterial strains is characterized in that a sample to be separated is subjected to domestication and enrichment culture, and is transferred to a culture medium taking polyethylene as a unique carbon source for culture for a period of time, and bacterial strains with polyethylene degradation capability are collected; the acclimatization and enrichment culture are performed in a mineral salt medium without a carbon source.
7. The method of claim 6, wherein the mineral salt medium without carbon source comprises: k (K) 2 HPO 4 ·3H 2 O 0.5-1.0g/L，KH 2 PO 4 0.5-1.0g/L，NH 4 NO 3 1.0g/L，NaCl 0.5g/L，MgSO 4 ·7H2O0.3-0.7g/L，FeSO4·7H 2 O 0.002g/L，ZnSO 4 ·7H 2 O 0.002g/L，MnSO 4 ·H 2 O 0.001g/L，CuSO 4 0.001g/L。
8. A method of degrading polyethylene and materials thereof, characterized in that the polyethylene plastic degrading strain of claim 1 is contacted with polyethylene or polyethylene material for a period of time to depolymerize the polyethylene.
9. The method of claim 8, wherein the polyethylene material is subjected to ultraviolet radiation pretreatment.
10. The method according to claim 9, wherein the ultraviolet irradiation treatment is to subject the polyethylene plastic article to ultraviolet irradiation of 275 to 200nm at 60 to 80 ℃ for at least 1d, or at least 2d, or at least 3d.
11. The method according to any one of claims 8 to 10, wherein the polyethylene plastic-degrading strain according to claim 1 is incubated at 28 to 32 ℃ for at least 10d in an environment containing said polyethylene plastic articles.
12. Use of a polyethylene plastic degrading strain according to claim 1, or a microbial preparation according to any one of claims 2-3, or a method according to any one of claims 8-11 for degradation of polyethylene plastic articles, recycling of polyethylene plastic, upgrade reconstruction of polyethylene plastic, and/or surface modification of polyethylene plastic.
CN202311836478.3A 2023-12-28 2023-12-28 Polyethylene plastic degrading bacterium screening method and application Pending CN117821314A (en)

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