A paraffin and polyethylene efficient degradation bacterium and its application

Abstract

The present invention belongs to the field of microorganisms and environmental protection technology, and specifically relates to a paraffin and polyethylene efficient degradation bacteria and its application. The ornithine Raoultella DY-2415 obtained by the present invention can not only effectively degrade liquid paraffin, but also has a good degradation effect on polyethylene plastics. It is also found that the strain has a certain degradation effect on polystyrene. The strain of the present invention is used to degrade polyethylene film. After two months, the plastic surface is severely damaged, obvious pits appear, and the weight loss rate reaches 8%, indicating that it has a good polyethylene degradation effect. The degradation strain of the present invention has the advantages of green environmental protection, low cost, and easy operation, and provides new resources and ideas for the treatment of paraffin cleaning and prevention and polyolefin plastic waste such as polyethylene, and therefore has good practical application value.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms and environmental protection, and specifically relates to a paraffin and polyethylene efficient degradation bacterium and application thereof. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Oil is one of the most important energy sources in modern society. The wax content of crude oil is high during oil production. Most crude oils have a wax content of more than 20%, and some even have a wax content of 40-50%. Most of them are long carbon chain wax molecules, which cause very serious waxing phenomenon, thus bringing serious impact on oil production. Therefore, it is particularly important to take necessary measures to remove and prevent wax. At present, hot washing (such as hot water washing or hot oil washing) is usually used, or chemical wax removal and prevention agents are used for wax removal and prevention. However, the former has a large workload and poor effect, and the use of hot water to wash wells will cause formation clay expansion and cause oil production blockage, and long-term hot washing will damage the structure of the oil well; while the use of chemical wax removal and prevention agents is relatively expensive, and will reduce the quality of crude oil, causing damage to the environment and being highly toxic to the human body.

[0004] Plastics are polymer compounds obtained by polymerization of organic monomers such as petroleum through addition polymerization or condensation polymerization. They are widely used in daily life due to their chemical stability, mechanical properties, durability and economy. However, plastics are hardly decomposed after disposal, and their continuous accumulation in the environment poses a serious ecological threat. Plastic pollution has become an environmental problem faced by the world. Polyethylene (PE) is one of the synthetic polymers with the highest production and usage in plastic products. Its basic structure is -[CH2-CH2] n -, due to its large relative molecular mass, strong hydrophobicity, high chemical bond energy, and low bioaccessibility, it is extremely difficult to degrade in the natural environment. At present, most of the methods for disposing of waste plastics are landfill, incineration, secondary processing, etc. However, these treatment methods are highly polluting to the environment, have high treatment costs, and are very likely to cause secondary pollution.

[0005] The inventors have found through research that there are relatively few studies on the biological treatment of paraffin wax and plastic wastes such as polyethylene, and effective bacterial resources are rare, which limits its further expansion and application. Summary of the invention

[0006] In view of the above-mentioned deficiencies of the prior art, the present invention provides a paraffin and polyethylene efficient degradation bacterium and its application. The present invention screened and obtained a new strain of Raoultella sp.DY-2415, which was proved by experiments to be not only capable of degrading liquid paraffin, but also had a good degradation effect on polyolefin plastics such as polyethylene and polystyrene, so it has good practical application value.

[0007] The present invention is achieved through the following technical solutions:

[0008] The first aspect of the present invention provides a strain of Raoultella sp. DY-2415, which has been sent to the China Center for Type Culture Collection (address: Wuhan University, Luojia Mountain, Wuchang, Wuhan City, Hubei Province), with a preservation date of July 4, 2022 and a preservation number of CCTCC NO: M 20221013.

[0009] The second aspect of the present invention provides a method for culturing the above-mentioned Raoultella ornithine, which comprises inoculating the Raoultella ornithine DY-2415 into a fermentation medium for fermentation culture.

[0010] The fermentation medium may be a common bacterial culture medium. In the present invention, the culture method is not specifically limited, and any conventional bacterial fermentation culture method may be used for culture.

[0011] The third aspect of the present invention provides a microbial agent containing the Raoultella ornithine DY-2415 or its fermentation product or metabolite.

[0012] The fourth aspect of the present invention provides the use of the above-mentioned Raoultella ornithine DY-2415 or the above-mentioned microbial agent in all or part of the following a)-b):

[0013] a) degrading paraffin or preparing a paraffin degradation agent;

[0014] b) Degrading plastics or preparing plastic degrading agents.

[0015] A fifth aspect of the present invention provides a method for degrading paraffin, which comprises applying the above-mentioned Raoultella ornithine DY-2415 and / or bacterial agent to a paraffin-containing environment.

[0016] Wherein, the paraffin is liquid paraffin.

[0017] A sixth aspect of the present invention provides a method for degrading plastics, the method comprising applying the above-mentioned Raoultella ornithine DY-2415 and / or bacterial agent to an environment containing plastics.

[0018] Wherein, the plastic is a polyolefin plastic, including but not limited to polyethylene plastic and polystyrene plastic.

[0019] Beneficial effects of one or more of the above technical solutions:

[0020] The ornithine Raoultella DY-2415 obtained by screening the above technical scheme can not only effectively degrade liquid paraffin, but also has a good degradation effect on polyethylene plastics. It is also found that the strain has a certain degradation effect on polystyrene. The strain of the present invention is used to degrade polyethylene film. After 2 months, the plastic surface is severely damaged, with obvious pits, and the weight loss rate reaches 8%, indicating that it has a good polyethylene degradation effect.

[0021] The above technical solution has the advantages of being green, environmentally friendly, low cost, and easy to operate. It provides new resources and ideas for the treatment of paraffin wax cleaning and prevention and the treatment of polyolefin plastic waste such as polyethylene, and therefore has good practical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0023] Figure 1 This is a colony morphology diagram of the polyethylene-degrading bacteria provided by the present invention;

[0024] Figure 2 This is a diagram showing the growth of the liquid paraffin-degrading strain of the present invention on an inorganic salt plate with liquid paraffin as the only carbon source;

[0025] Figure 3 The gas phase diagram of alkane compounds of the liquid paraffin-degrading strain of the present invention before and after growth in a liquid culture medium;

[0026] Figure 4 A growth curve diagram of the polyethylene degrading bacteria provided by the present invention;

[0027] Figure 5 A phylogenetic tree of polyethylene-degrading bacteria provided by the present invention;

[0028] Figure 6 The changes of functional groups on the surface of polyethylene film before and after degradation by the polyethylene degrading bacteria provided by the present invention;

[0029] Figure 7 The figures are scanning electron microscope images of polyethylene film and polystyrene film after being degraded by the polyethylene degrading bacteria provided by the present invention for 60 days; A is a blank control; B is a polystyrene film; and C is a polyethylene film. DETAILED DESCRIPTION

[0030] It should be noted that the following detailed descriptions are all illustrative and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0031] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components and / or combinations thereof. It should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the embodiments of the present invention are intended to describe specific embodiments, rather than to limit the scope of protection of the present invention.

[0032] As mentioned above, there are relatively few studies on the biological treatment of both paraffin and plastic wastes such as polyethylene, and effective bacterial resources are rare, which limits its further expansion and application.

[0033] In view of this, the present invention screened and obtained a new strain of Raoultella sp. DY-2415, which was proved by experiments to be not only capable of degrading liquid paraffin, but also had a good degradation effect on polyolefin plastics such as polyethylene and polystyrene.

[0034] Specifically, in a typical embodiment of the present invention, a strain of Raoultella sp. DY-2415 is provided. The strain has been sent to the China Type Culture Collection (address: Wuhan University, Luojia Mountain, Wuchang, Wuhan City, Hubei Province), with a preservation date of July 4, 2022 and a preservation number of CCTCC NO: M 20221013.

[0035] In another specific embodiment of the present invention, a method for culturing the above-mentioned Raoultella ornithine is provided, and the culturing method comprises inoculating the Raoultella ornithine DY-2415 into a fermentation medium for fermentation culture.

[0036] The fermentation medium may be a common bacterial culture medium. In the present invention, the culture method is not specifically limited, and any conventional bacterial fermentation culture method may be used for culture.

[0037] In another specific embodiment of the present invention, a microbial agent is provided, which contains the Raoultella ornithine DY-2415 or its fermentation product or its metabolite.

[0038] In the present invention, the term "fermented product" is used to refer to a fermented product. The corresponding fermented product may be a liquid obtained from the process of fermenting and culturing Raoultella ornithine DY-2415, and therefore, may also be referred to as a fermentation liquid; the liquid may contain bacteria (bacteria), but does not necessarily need to contain bacteria. The liquid preferably contains metabolites produced by Raoultella ornithine DY-2415 of the present invention.

[0039] Also, in an embodiment of the present invention, the fermentation liquid or culture liquid containing the bacteria is centrifuged, filtered, sedimented or other means known in the art to separate the bacterial cells grown in the fermentation liquid or culture liquid from the liquid, and the liquid remaining after removing the bacterial cells is the "supernatant", and in the present invention, the supernatant contains the extracellular metabolites of Raoultella ornithine DY-2415. In an embodiment of the present invention, the bacterial agent may also include the supernatant.

[0040] And, in an embodiment of the present invention, the fermentation liquid or culture liquid containing the bacteria is centrifuged, filtered, sedimented or other means known in the art to separate the bacterial cells grown in the fermentation liquid or culture liquid from the liquid to obtain the bacteria, and the bacteria can be crushed to obtain the bacterial crushed material, and the crushing method can be ultrasound (such as ice bath ultrasonic cell crushing) or other means known in the art, or, further, the bacterial crushed material is centrifuged to collect the supernatant, and the supernatant is recorded as a cell-free extract, and in the present invention, the bacterial crushed material or cell-free extract contains the intracellular metabolites of Raoultella ornithine DY-2415. In an embodiment of the present invention, the bacterial agent may also include the bacterial crushed material or cell-free extract.

[0041] Furthermore, in the embodiments of the present invention, in order to facilitate storage and transportation, improve the survival rate of the strain, etc., the bacterial agent can also be solid, and more preferably a freeze-dried powder. That is, the above-mentioned Raoultella ornithine DY-2415 or its fermentation product or its metabolite is further freeze-dried to obtain the freeze-drying technology (including vacuum freeze-drying technology) can be carried out by conventional methods, which will not be repeated here.

[0042] In another specific embodiment of the present invention, the microbial agent may further include excipients acceptable to the agent.

[0043] In another specific embodiment of the present invention, the auxiliary material is selected from one or more of a dispersant, a wetting agent, a disintegrant, a binder, an antifreeze agent, a thickener, a filler and a solvent. The present invention has no special restrictions on the source of the auxiliary materials acceptable to the bacterial agent, and generally commercial products can be used.

[0044] The dispersant is an anionic dispersant and / or a nonionic dispersant, which can be selected from one or more of sodium lignin sulfonate, sodium naphthalene sulfonate formaldehyde condensate, sodium methylene bisnaphthalene sulfonate, formaldehyde condensate sulfate, polycarboxylate, alkylphenol polyoxyethylene phosphate and fatty acid polyoxyethylene ester.

[0045] The wetting agent can be selected from one or more of sodium dodecyl sulfate, sodium dodecylbenzene sulfonate, saponin powder, soapberry powder, tea dregs powder and lakai powder BX.

[0046] The disintegrant may be selected from one or more of bentonite, ammonium sulfate, aluminum chloride, urea, magnesium chloride and glucose.

[0047] The binder may be selected from one or more of starch, diatomaceous earth, cyclodextrin, rosin, carboxymethyl cellulose, carboxyethyl cellulose and carboxymethyl cellulose salt.

[0048] The antifreeze agent may be selected from one or more of sorbitol, ethylene glycol, polyethylene glycol, propylene glycol, glycerol, urea and sodium chloride.

[0049] The thickener may be selected from one or more of gelatin, xanthan gum, polyethylene glycol and polyvinyl alcohol.

[0050] The filler may be selected from one or more of light calcium carbonate, diatomaceous earth, bentonite, attapulgite and white carbon black.

[0051] The solvent may be selected from water (preferably deionized water) or methyl oleate.

[0052] In another specific embodiment of the present invention, the use of the above-mentioned Raoultella ornithine DY-2415 or its fermentation product or its metabolite in all or part of the following a)-b) is also within the scope of protection of the present invention:

[0053] a) degrading paraffin or preparing a paraffin degradation agent;

[0054] b) Degrading plastics or preparing plastic degrading agents.

[0055] in,

[0056] In said a), the paraffin includes liquid paraffin;

[0057] In b), the plastic is a polyolefin plastic, including but not limited to polyethylene plastic and polystyrene plastic.

[0058] In another specific embodiment of the present invention, a method for degrading paraffin is provided, the method comprising applying the above-mentioned Raoultella ornithine DY-2415 and / or bacterial agent to a paraffin-containing environment.

[0059] In another specific embodiment of the present invention, the paraffin is liquid paraffin.

[0060] In another specific embodiment of the present invention, a method for degrading plastic is provided, the method comprising applying the above-mentioned Raoultella ornithine DY-2415 and / or bacterial agent to an environment containing plastic.

[0061] In another specific embodiment of the present invention, the plastic is a polyolefin plastic, including but not limited to polyethylene plastic and polystyrene plastic.

[0062] In order to better illustrate the purpose, technical scheme and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments. The raw materials and reagents used in the embodiments of the present invention are all commercially available products unless otherwise specified, and the experimental methods and instruments used are all conventional laboratory techniques.

[0063] Example 1 Experiment on the degradation of liquid paraffin by liquid paraffin-degrading strain DY24-15

[0064] (1) Screening of liquid paraffin-degrading strains

[0065] In this example, liquid paraffin was used for primary screening, and screening and culturing were performed in an inorganic salt medium using liquid paraffin as the sole carbon source. After three transfers, the strains were isolated using a dilution plate method, and the isolated strains were purified by multiple streaking separations.

[0066] The liquid inorganic salt culture medium formula for separation and screening is: 1g / L (NH4) 2SO4, 0.5g / L KH2PO4, 1.5g / L Na2HPO4, 1g / L NaCl, 0.2g / L MgSO4.7H2O, and 1.5% agar is added to form a solid culture medium. Through the above separation and screening work, after multiple separation and purification, a strain that can use ultraviolet irradiated polyethylene as a carbon source and energy source for growth was obtained, named DY-2415. This strain was isolated from petroleum-contaminated soil. Its colonies on the nutrient agar plate are round, smooth, neatly edged, and light yellow. Figure 1 shown.

[0067] Prepare an inorganic salt solid plate and pour it. After it dries, spread 100 μL of liquid paraffin on it. After it dries, streak the strain for screening. Then culture it in a 37°C incubator for 3 days and observe the growth of the strain. It was found that the strain DY-2415 can grow on the solid culture with liquid paraffin as the only carbon source, indicating that the bacteria can use liquid paraffin as a carbon source and energy for growth metabolism.

[0068] We then cultured DY-2415 in a liquid culture medium with liquid paraffin as the sole carbon source, and set up a control group, that is, liquid paraffin was added without inoculation. After 1 week of culture, the bacterial liquid was extracted with dichloromethane, and the organic phase was taken for gas chromatography detection. The results are as follows: Figure 3 As shown, compared with the control group, the alkane content of the bacterial liquid in the experimental group was significantly lower than that in the control group, which once again confirmed that the bacteria can degrade liquid paraffin.

[0069] (2) Molecular biological identification of strain DY-2415

[0070] The universal primers 27F and 1492R were used as amplification primers to perform PCR amplification on the single strain isolated above.

[0071] 16S rDNA sequence primer 27F: 5'-AGAGTTTGATCMTGGCTCAG-3' (SEQ ID NO. 1), 1492R: 5'-TACGGYT ACCTTGTTACGACTT-3' (SEQ ID NO. 2).

[0072] The amplified product was tested by electrophoresis and sequenced by Qingdao Qingke Biotechnology Co., Ltd. The 16S rDNA sequence is shown below. The obtained sequence was compared with the existing sequence in the NCBI database by BLAST analysis, and strains with similar homology were selected to construct a phylogenetic tree using MEGA 11.0 software. The 16S rDNA sequence was compared on NCBI, and the results showed that the similarity with Raoultella ornithine was 100%, and the evolutionary distance was relatively close. The phylogenetic tree of the strain is shown below. Figure 2 As shown, combined with the physiological and biochemical characteristics of the strain, it was preliminarily identified as Raoultella sp. and named Raoultella sp. DY-2415. The strain was sent to the China Center for Type Culture Collection (Wuhan University) for preservation, with the preservation number CCTCC NO: M 20221013 and the preservation date of July 4, 2022.

[0073] The 16S rDNA sequence of the strain is as follows:

[0074]

[0075] Example 2 Degradation experiment of polyethylene film by polyethylene-degrading strain DY-2415

[0076] Preparation of UVPE film: Place the purchased PE film in an aging box with a UVB313 nm ultraviolet lamp for irradiation. After 20 days of irradiation, take it out and soak it in 75% ethanol for cleaning, and dry it in a clean bench for later use.

[0077] Pick a single colony into 5 mL LB liquid medium, culture overnight at 37°C, 220 rpm, collect the cells by centrifugation (7000 rpm, 10 min), wash three times with inorganic salt medium to remove the residual LB medium, and resuspend the cells in 1 mL inorganic salt medium to prepare a cell suspension.

[0078] The cell suspension was added into an inorganic salt culture medium with UVPE film as the sole carbon source, and cultured at 37°C and 120 rpm for 60 days to analyze the changes in functional groups, morphology and hydrophobicity on the surface of the plastic film.

[0079] Treatment of plastic film after culture: The plastic after culture was fixed in 2.5% glutaraldehyde, then washed with 1×PBS solution and ethanol of different concentration gradients, and then dried using a critical point dryer. After gold spraying, it was observed by scanning electron microscopy. The plastic film after culture was ultrasonically cleaned with 1% SDS solution for 20 minutes, then ultrasonically cleaned with deionized water for 20 minutes, and finally ultrasonically cleaned with 75% ethanol for 20 minutes. The functional group changes on the surface of the film were measured using a Fourier transform infrared spectrometer with a scanning wavelength of 600-4000cm -1 , resolution 4cm -1 , scanning times 32 times. The mass was weighed before culture, and the mass was weighed after cleaning and drying after culture, and the weight loss rate was calculated using the formula: (mass before culture - mass after culture) / mass before culture × 100%.

[0080] After 60 days of cultivation, the weight loss rate reached 8%, which was nearly 2 times higher than the weight loss rate of 4.3% of Raoultella ornithinolytica MP-1 (see CN201910743229.7) after 60 days. Therefore, the strain Raoultellasp.DY-2415 in the present invention has a more obvious degradation effect on polyethylene plastics.

[0081] Depend on Figure 6 As shown in the figure, after 60 days of cultivation, in addition to the carbonyl peak of the original UVPE film, hydroxyl -OH (1000cm -1 and 3500cm -1), the appearance of these new functional groups indicates the biodegradation of UVPE film; compared with Raoultella ornithinolytica MP-1, the degradation effect is more obvious. MP-1 only shows carbonyl peak after 60 days of cultivation, while DY-2415 in the present invention shows obvious hydroxyl peak, indicating that the oxidation effect of polyethylene plastic is more obvious. Figure 7 As shown, after 60 days of cultivation, a biofilm was formed on the surface of the UVPE membrane. Compared with the blank control membrane, the surface was cracked and rough. Compared with MP-1, the adhesion effect and damage effect were more obvious.

[0082] Therefore, compared with the strain Raoultella ornithinolytica MP-1, whether from the weight loss rate or the results of infrared spectrum and scanning electron microscopy, the degradation effect of Raoultella sp.DY-2415 in the present invention is better than that of the strain in the above patent, and the strain in the present invention is screened from petroleum-contaminated soil, has a degradation effect on liquid paraffin, that is, it also has a degradation effect on some alkane compounds, and may also have a certain degradation effect on other polyolefin plastics except polyethylene, so the degradation substrate spectrum of Raoultella sp.DY-2415 in the present invention is wider.

[0083] It should be noted that the above examples are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention is described in detail with reference to the given examples, those skilled in the art may modify or replace the technical solution of the present invention as needed without departing from the spirit and scope of the technical solution of the present invention.

Claims

1. A strain of Raoultella ornithine Raoultella sp. DY-2415, the strain has been sent to the China Center for Type Culture Collection, the preservation date is July 4, 2022, and the preservation number is CCTCC NO: M 20221013.

2. The method for culturing Raoultella ornithine according to claim 1, characterized in that: The culturing method comprises the steps of inoculating the Raoultella ornithine DY-2415 into a fermentation medium for fermentation culturing.

3. A microbial agent, characterized in that: It contains the Raoultella ornithine DY-2415 according to claim 1.

4. The microbial agent according to claim 3, characterized in that: The microbial agent also includes auxiliary materials acceptable to the agent.

5. The microbial agent according to claim 4, characterized in that: The auxiliary material is selected from one or more of a dispersant, a wetting agent, a disintegrant, a binder, an antifreeze agent, a thickener, a filler and a solvent.

6. Use of Raoultella ornithine DY-2415 according to claim 1 or the microbial agent according to any one of claims 3 to 5 in all or part of the following a) to b): a) Degrading paraffin or preparing paraffin degradation agents; b) Degrading plastics or preparing plastic degradation agents; in, The paraffin is liquid paraffin; and the plastic is polyethylene plastic or polystyrene plastic.

7. A method for degrading paraffin, characterized in that: The method comprises applying the Raoultella ornithine DY-2415 of claim 1 and / or the microbial agent of any one of claims 3 to 5 to a paraffin-containing environment; Wherein, the paraffin is liquid paraffin.

8. A method for degrading plastics, characterized in that: The method comprises applying the Raoultella ornithine DY-2415 of claim 1 and / or the microbial agent of any one of claims 3 to 5 to an environment containing plastic; Wherein, the plastic is polyethylene plastic or polystyrene plastic.

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