CN112553108B - Bacillus brevis and application thereof in degradation of polyurethane - Google Patents
Bacillus brevis and application thereof in degradation of polyurethane Download PDFInfo
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Abstract
The invention belongs to the technical field of biological treatment, and particularly discloses a brevibacillus brevis and application thereof in degradation of polyurethane. The strain of Brevibacillus brevis is classified and named as Brevibacillus brevis (Brevibacillus sp.), the strain name is P10, the strain is preserved in China center for type culture collection, and the preservation number of the strain is CCTCC NO: m2020626, the preservation date is 10 months and 23 days in 2020. The degradation strain provided by the invention not only can degrade PU simulant Impranil DLN, but also shows good degradation capability of real polyurethane plastic film. This is not available from many known strains capable of degrading Impranil DLN. In addition, the strain P10 has low cost of experimental consumables and green and efficient experimental technology, and can bring higher benefit for the development of a polyurethane biodegradation biotechnology process.
Description
Technical Field
The invention belongs to the technical field of biological treatment, and particularly relates to a brevibacillus brevis and application thereof in degradation of polyurethane.
Background
Plastics are used in large quantities in our modern society and global productivity has been increasing for decades. In 2015, polyurethane was about 350 ten thousand tons, which is the fifth most demanding synthetic polymer in europe. Polyurethanes are all known as polyurethanes, known by the english name Polyurethane and may be abbreviated as PU. Common precursors for the synthesis of polyurethanes are polyisocyanates and polyols and additives such as catalysts, crosslinkers and chain extenders. Although forming urethane bonds with polyisocyanates, polyols also contain ether or ester bonds, resulting in polyether or polyester polyurethanes, respectively. By varying the type and ratio of polyol and isocyanate, PUs of various properties can be produced. Therefore, the polyurethane has various applications, and is mainly used as an insulating material in the industrial fields of medicine, automobiles and the like.
Because PU is difficult to degrade under natural conditions, the existing recovery treatment methods such as chemical degradation and physical incineration have the problems of slowness, limitation, ecological environment damage and the like, so that the method for degrading polyurethane waste by utilizing microorganisms as a green and efficient solution becomes a hot spot.
Most PU products have high hydrophobicity, and are not favorable for the attachment and utilization of microorganisms. Impranil DLN is a commercial aqueous polyester polyurethane dispersion, is in a milky white liquid state, and can be used as a structure simulator to screen PU plastic degrading bacteria. Although some fungi and bacteria capable of degrading DLN, such as Cladosporium sp, aspergillus sp and Penicillium sp, have been reported, the specific types of strains capable of degrading real PU plastics are few, and PU plastic degrading microorganisms are still in the excavation stage. Therefore, PU simulacrum and real polyurethane plastic are combined as substrates to research the degradation effect of the Impranil DLN degradation strain on the PU simulacrum DLN degradation strain, and detection methods such as mass loss, esterase activity, fourier transform infrared spectroscopy, scanning electron microscope and the like are adopted to provide evidence for the biological action of the polyurethane plastic degradation strain. The degradation bacterial strain obtained by the research screening can enable the development of the biological technology process of the polyurethane biodegradation to be more valuable.
Disclosure of Invention
The invention aims to: the invention aims to solve the technical problem of providing a strain of brevibacillus brevis aiming at the defects of the prior art.
The invention also aims to solve the technical problem of providing the application of the brevibacillus brevis in degrading polyurethane.
In order to solve the technical problem, the invention discloses a Brevibacillus brevis which is classified and named as Brevibacillus brevis (Brevibacillus sp.), the strain name is P10, the Brevibacillus brevis is preserved in China center for type culture collection, wuhan university in Wuhan, china, the strain preservation number is CCTCC NO: m2020626, having a preservation date of 10/23/2020, belongs to the strictly aerobic Brevibacillus genus.
Wherein, the Brevibacillus brevis (Brevibacillus sp.) P10 is separated from soil near Nantong sponge manufacturing plant, and is screened, purified and cultivated on selective MSM agar plate containing Impranil DLN (1%), and is obtained by morphology and molecular biology, the strain is determined to be Brevibacillus brevis, and has 99.43 percent of homology with Brevibacillus formosus (NR _ 040979.1); the main morphological characteristics of the Brevibacillus brevis (Brevibacillus sp.) P10 are shown in figure 1: the bacterial colony is flat, milky oval, wet and smooth in surface, neat in edge and opaque. A clear degradation ring can be formed on a selective MSM agar plate containing Impranil DLN (1%); the 16S rRNA sequence of the Bacillus brevis (Brevibacillus sp.) P10 is shown in a sequence table SEQ ID No.1, the 16S rRNA sequence of the strain P10 is compared with the 16S rRNA sequence in a GenBank database in a similarity manner, and phylogenetic tree construction is carried out by adopting BioEdit and MEGA software (figure 2).
A microbial liquid comprising the Bacillus brevis or a fermentation liquid comprising the Bacillus brevis and the like is also within the protection scope of the invention.
The preparation method of the microbial strain liquid comprises the steps of carrying out shake flask culture fermentation on the brevibacillus brevis, and specifically, selecting a single colony from a DLN (DLN) plate after P10 strain purification, inoculating the single colony into 5mL of LB (LB) culture medium, and carrying out oscillation culture at 30-37 ℃ to obtain the microbial strain liquid; preferably, the culture temperature is 37 ℃ and the culture time is 12h.
The application of the brevibacillus brevis in degrading the waterborne polyurethane (imprianil DLN) is also within the protection scope of the invention.
The application of the microbial solution in degrading Impranil DLN is also within the protection scope of the invention.
Wherein, the application comprises that the microbial strain liquid is inoculated in an LB liquid culture medium and cultured to OD 600 0.5-0.6, adding waterborne polyurethane, and continuing to culture and degrade.
Wherein the culture is shaking culture at 30-37 ℃; preferably, the culture is at 37 ℃,180rpm culture.
The application of the brevibacillus brevis in degrading polyurethane is also within the protection scope of the invention.
The application of the microbial liquid in the degradation of polyurethane is also within the protection scope of the invention.
The application is that the commercial polyurethane film after sterilization is placed on an MSM-agarose flat plate, the microbial inoculum is coated on the polyurethane film, and the polyurethane plastic film can be directly degraded after culture at 30-37 ℃.
Wherein the dosage of the microbial inoculum is 10% (mu L/m) 2 )。
Has the advantages that: compared with the prior art, the invention has the following advantages:
(1) Due to the problems of large material energy consumption, influence on ecological environment and the like in the chemical degradation and physical incineration treatment of polyurethane plastics, the degradation strain provided by the invention can degrade a PU analogue Impranil DLN, shows good degradation capability of a polyurethane real plastic film, and can enable the quality loss of the film to be as high as 9.09% within 2 weeks in a polyurethane plastic film degradation experiment. This is not available in many known strains capable of degrading Impranil DLN, and therefore the present invention shows that this strain has a long-term value in the fields of polyurethane plastics contamination and bioremediation.
(2) The strain P10 has low cost of experimental consumables and green and efficient experimental technology, and brings higher benefit for the development of a polyurethane biodegradation biotechnology process.
Drawings
The foregoing and/or other advantages of the invention will become further apparent from the following detailed description of the invention when taken in conjunction with the accompanying drawings.
FIG. 1 shows the colony morphology of strain P10 on selective MSM agar plates containing Impranil DLN (1%).
FIG. 2 is a phylogenetic tree of strain P10 and related species 16S rRNA sequences.
FIG. 3 shows the change in extracellular supernatant esterase activity of strain P10 in degrading Impranil DLN.
FIG. 4 shows the change in functional groups after degradation of Impranil DLN by strain P10 for 5 days: (ii) (a) FTIR spectra of sterile treatment controls; (b) FTIR spectrum 5 days after addition of strain P10 to degrade Impranil DLN.
FIG. 5 is a graph of the mass loss of PU films degraded by strain P10 over 8 weeks.
FIG. 6 is an electron micrograph of PU thin film degraded by strain P10 in 8 weeks.
Detailed Description
The experimental methods described in the following examples are all conventional methods unless otherwise specified; the reagents and materials are commercially available, unless otherwise specified.
Example 1 isolation and identification of Strain P10
Inoculating 1-2g of soil thin extract from the periphery of Nantong sponge manufacturing plant into 50mL of enrichment medium containing 0.2% of Impranil DLN, and culturing at 37 deg.C for 180 r.min -1 And (5) performing transfer culture. The enrichment bacterial liquid of Impranil DLN is treated according to the proportion of 10 3 -10 6 After double dilution, each was plated on selective MSM agar plates containing Impranil DLN (1%) and cultured in an inverted state at 37 ℃ for 4-5 days. Selecting a bacterial colony with an obvious hydrolysis transparent ring, and marking a line on three areas of a blank MSM agar plate, wherein a single bacterial colony is a degradation strain capable of utilizing Impranil DLN; wherein the enrichment medium comprises the following components: 10g/L tryptone, 10g/L sodium chloride, 5g/L yeast powder, 0.2% (V/V) Impranil DLN and distilled water to 1000mL; the selective MSM culture medium comprises the following components: 1.0g/L of ammonium sulfate, 1.0g/L of sodium chloride, 0.5g/L of monopotassium phosphate, 1.5g/L of dipotassium phosphate and 0.7g/L of magnesium sulfate heptahydrate are prepared, the pH value is adjusted to 7.0, 15.0-20.0g/L of agar is added into a solid culture medium, sterilization is carried out at 121 ℃ for 20min, and the mixture is mixed after sterilization by Impranil DLN, wherein the concentration is 1% (V/V).
The colony of strain P10 on selective MSM agar plates containing Impranil DLN (1%) was flat and milky white, with smooth surface, oval spores, and a distinct hydrolysis ring on the plate (FIG. 1). Single colonies on the plate were picked and inoculated in 5mL LB tubes at 37 ℃ and 180 r.min -1 Culturing in a shaking table for 12h. The cultured bacterial solution was used as a carrier, and the mixture was cultured using a primer 27F:5 '-AGAGTTTGATCCTGGCTCAG-3' and 1492R: 5 '-GGTTACCTTGTTACGACTT-3' amplifying the 16S rRNA sequence of the P10 strain, sequencing the obtained amplified fragments, comparing the 16S rRNA sequence by NCBI database, constructing a phylogenetic tree of the strain P10 and related species 16S rRNA sequence, identifying the strain P10 as Brevibacillus at molecular level (figure 2), wherein the nucleotide sequence of the 16S rRNA is shown as SEQ ID No.1 in the sequence table. The obtained strains are sent to China Center for Type Culture Collection (CCTCC) with the preservation numbers: CCTCC NO: M2020626P 10.
Example 2: degradation characteristics of strain P10 on DLN
The presence of esterase activity in the degradation of DLN by strain P10 was presumed from the hydrolysis loop of strain P10 on selective MSM agar plates of Impranil DLN (1%). A single colony was picked from a DLN plate purified from the P10 strain, inoculated in a 5mL LB tube, and subjected to shake culture at 37 ℃ for 12 hours to obtain a P10 bacterial solution. Transferring the P10 bacterial liquid into 100mL LB liquid culture medium according to the inoculum size of 1% in volume ratio, at 37 ℃ and 180 r.min -1 Culturing for 1-3h under the condition, sampling every 1h to determine OD 600nm When OD is reached 600nm About 0.5 to 0.6, 1% by volume of DLN was added to induce the production of enzyme by strain P10, and the enzyme was degraded by culturing, and cultured at 30 ℃ and 180rpm with shaking, and the esterase activity produced by strain P10 was measured every 24 hours.
And (3) measuring the esterase activity: sampling fermentation liquor for degrading DLN by the strain P10 every 24h, and carrying out esterase activity test; wherein the test reaction system (2 mL) is 880 uL of 50mM phosphate buffer solution, 200 uL of enzyme extract, 40 uL of 10 mM isopropanol solution of p-NPB, the reaction is carried out for 30min at 37 ℃, the absorbance is measured at the wavelength of 410nm, and a blank control is arranged; definition of esterase activity: at 37 ℃ the esterase catalyzes p-nitrophenol butyrate (p-NPB) per ml of culture medium, and the amount of p-nitrophenol (p-NP) produced per minute is defined as one enzyme activity unit. It was found that not only esterase was present but also esterase activity was as high as 1.08U/mL at 72h (FIG. 3).
Respectively freeze-drying the aseptically processed control fermentation supernatant and the fermentation supernatant of the fifth day of degrading DLN by the strain P10 by using a freeze dryer, wherein the freeze-dried solid is 2500-800 cm -1 FTIR spectrum analysis was performed over the range, 1735cm in test sample (b) compared to control (a) as shown in FIG. 4 -1 Representing the ester carbonyl function, disappearance of the absorption peak indicates hydrolysis of the ester bond. In addition, no significant change in other peaks indicates that the key chemical bond amide bond in DLN is relatively stable and does not undergo degradation. However, most of the literature on PU degradation mechanism occurs in the ester bond cleavage of the polyol part, which is consistent with the results of the present invention.
It can be seen that the strain P10 not only changes along with the DLN functional group in the degradation process of DLN, but also produces esterase, and has a certain synergistic effect.
Example 3: degradation of real polyurethane plastic film by DLN degrading bacteria P10
A single colony was picked from a DLN plate purified from the P10 strain, inoculated in a 5mL LB tube, and subjected to shake culture at 37 ℃ for 12 hours to obtain a P10 bacterial solution. The sterilized commercial polyurethane film was placed on an MSM-agarose plate, and 200. Mu.L of LB-cultured P10 bacterial solution was uniformly applied to the plate, and the plate was cultured at 37 ℃ for 8 weeks, and the degree of degradation of the film by the strain P10 was observed by sampling every 2 weeks. Macroscopically, the degradation effect is distinguished by comparing the mass loss of the sample before and after the control treatment; and (4) carrying out SEM analysis on the gold sprayed on the surface of the film on a microscopic scale. It was found that the PU film achieved 9.09% mass loss at week 2 compared to the sterile-treated control, and that the mass loss stabilized to 15.1% at week 8, although the rate of mass loss was slowed at week 4,6,8 (fig. 5). The strain P10 can directly utilize the PU real plastic film and achieve obvious degradation effect. Similarly, changes in the film surface as shown by scanning with an electron microscope correspond to weekly mass loss. As can be seen in fig. 6, the film surface at 2 weeks was clearly perforated and cracked compared to the entire surface of the aseptically processed film; the holes and cracks are further deepened at 4 weeks, 6 weeks and 8 weeks, so that the degradation strain P10 can be determined to have a degradation effect not only on the structural simulant Impranil DLN, but also on real solid PU, a solid theoretical basis is laid for the research of PU biodegradation, and the development of polyurethane biodegradation and recycling biotechnology is more valuable.
The invention provides a strain of brevibacillus brevis and a thought and a method for application thereof in degrading polyurethane, and a plurality of methods and ways for realizing the technical scheme are provided, the above description is only a preferred embodiment of the invention, and it should be noted that, for a person skilled in the art, a plurality of improvements and embellishments can be made without departing from the principle of the invention, and the improvements and embellishments should be regarded as the protection scope of the invention. All the components not specified in the present embodiment can be realized by the prior art.
Sequence listing
<110> Nanjing university of industry
<120> Bacillus brevis and application thereof in degradation of polyurethane
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1432
<212> DNA
<213> Brevibacillus brevis (Brevibacillus sp.)
<400> 1
atgctagcat gatatacgtg cagtcgagcg agtctcttcg gaggctaccg gcggacgggt 60
gagtaacacg taggcaacct gcctctcaga ctgggataac atagggaaac ttatgctaat 120
accggatagg tttttggatc gcatgatccg aaaagaaaag atggcttcgg ctatcactgg 180
gagatgggcc tgcggcgcat tagctagttg gtggggtaac ggcctaccaa ggcgacgatg 240
cgtagccgac ctgagagggt gaccggccac actgggactg agacacggcc cagactccta 300
cgggaggcag cagtagggaa ttttccacaa tggacgaaag tctgatggag caacgccgcg 360
tgaacgatga aggtcttcgg attgtaaagt tctgttgtta gggacgaata agtaccgttc 420
gaatagggcg gtaccttgac ggtacctgac gagaaagcca cggctaacta cgtgccagca 480
gccgcggtaa tacgtaggtg gcaagcgttg tccggattta ttgggcgtaa agcgcgcgca 540
ggcggctatg taagtctggt gttaaagccc ggggctcaac cccggttcgc atcggaaact 600
gtgtagcttg agtgcagaag aggaaagcgg tattccacgt gtagcggtga aatgcgtaga 660
gatgtggagg aacaccagtg gcgaaggcgg ctttctggtc tgtaactgac gctgaggcgc 720
gaaagcgtgg ggagcaaaca ggattagata ccctggtagt ccacgccgta aacgatgagt 780
gctaggtgtt gggggtttca ataccctcag tgccgcagct aacgcaataa gcactccgcc 840
tggggagtac gctcgcaaga gtgaaactca aaggaattga cgggggcccg cacaagcggt 900
ggagcatgtg gtttaattcg aagcaacgcg aagaacctta ccaggtcttg acatcccgct 960
gatcgctctg gagacagagc ttcccttcgg ggcagcggtg acaggtggtg catggttgtc 1020
gtcagctcgt gtcgtgagat gttgggttaa gtcccgcaac gagcgcaacc cttatcttta 1080
gttgccagca ttcagttggg cactctagag agactgccgt cgacaagacg gaggaaggcg 1140
gggatgacgt caaatcatca tgccccttat gacctgggct acacacgtgc tacaatggtt 1200
ggtacaacgg gatgctacct cgcgagagga cgccaatctc ttaaaaccaa tctcagttcg 1260
gattgtaggc tgcaactcgc ctacatgaag tcggaatcgc tagtaatcgc ggatcagcat 1320
gccgcggtga atacgttccc gggccttgta cacaccgccc gtcacaccac gggagtttgc 1380
aacacccgaa gtcggtgagg taaccgcaag gagccagccg ccgcactgag gt 1432
Claims (3)
1. Application of a strain of brevibacillus brevis or a bacterial liquid containing the brevibacillus brevis in degrading waterborne polyurethane, which is classified and named as brevibacillus brevis (Brevibacillus brevis)Brevibacillussp.), the name of the strain is P10, which is preserved in ChinaThe preservation number of the strain is CCTCC NO: m2020626, the preservation date is 10 months and 23 days in 2020;
the application comprises the step of inoculating a bacterial liquid containing the brevibacillus brevis into an LB liquid culture medium to culture to OD 600 Adding waterborne polyurethane into the mixture, and continuously culturing and degrading the mixture, wherein the content of the waterborne polyurethane is 0.5 to 0.6 percent; the culture is shaking culture at 30 to 37 ℃.
2. The use according to claim 1, wherein the method for preparing the bacterial liquid comprising the brevibacillus brevis comprises the steps of inoculating the brevibacillus brevis in an LB culture medium according to claim 1, and carrying out shake culture at 30-37 ℃.
3. Application of a strain of brevibacillus brevis or a bacterial liquid containing the brevibacillus brevis in degradation of polyurethane, which is classified and named as brevibacillus brevis (A)Brevibacillussp.), the strain name is P10, the strain is preserved in China center for type culture Collection, and the preservation number of the strain is CCTCC NO: m2020626, the preservation date is 10 months and 23 days in 2020;
the application is that the bacterium liquid containing the brevibacillus brevis is coated on polyurethane and cultured at 30-37 ℃, and then the bacterium liquid can be degraded.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2005060530A (en) * | 2003-08-12 | 2005-03-10 | Nakagawa Chem:Kk | Method for degrading polymer |
| CN109762744A (en) * | 2019-03-01 | 2019-05-17 | 中国科学院昆明植物研究所 | Fungal bacterial strain and its cultural method and purposes for degradable polyurethane plastics |
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| AU2014252165A1 (en) * | 2013-04-11 | 2015-11-05 | B.G. Negev Technologies Ltd. | Compositions and methods for biodegrading plastic |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2005060530A (en) * | 2003-08-12 | 2005-03-10 | Nakagawa Chem:Kk | Method for degrading polymer |
| CN109762744A (en) * | 2019-03-01 | 2019-05-17 | 中国科学院昆明植物研究所 | Fungal bacterial strain and its cultural method and purposes for degradable polyurethane plastics |
Non-Patent Citations (3)
| Title |
|---|
| 塑料降解与稳定化(Ⅴ):生物降解与生物稳定(上);吴茂英;《塑料助剂》;20110420(第02期);全文 * |
| 聚氨酯的生物降解研究进展;闫华等;《应用与环境生物学报》;20180413(第05期);全文 * |
| 降解聚氨酯用微生物的驯化与筛选;于俊林等;《天津工业大学学报》;20060430(第02期);全文 * |
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