CN113801822A - Bacillus vallismortis YZS-C10 and application thereof - Google Patents
Bacillus vallismortis YZS-C10 and application thereof Download PDFInfo
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- CN113801822A CN113801822A CN202111151301.0A CN202111151301A CN113801822A CN 113801822 A CN113801822 A CN 113801822A CN 202111151301 A CN202111151301 A CN 202111151301A CN 113801822 A CN113801822 A CN 113801822A
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Images
Classifications
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B29—WORKING OF PLASTICS; WORKING OF SUBSTANCES IN A PLASTIC STATE IN GENERAL
- B29B—PREPARATION OR PRETREATMENT OF THE MATERIAL TO BE SHAPED; MAKING GRANULES OR PREFORMS; RECOVERY OF PLASTICS OR OTHER CONSTITUENTS OF WASTE MATERIAL CONTAINING PLASTICS
- B29B17/00—Recovery of plastics or other constituents of waste material containing plastics
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
- Y02W30/00—Technologies for solid waste management
- Y02W30/50—Reuse, recycling or recovery technologies
- Y02W30/62—Plastics recycling; Rubber recycling
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- Engineering & Computer Science (AREA)
- Environmental & Geological Engineering (AREA)
- Mechanical Engineering (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention provides bacillus vallismortis YZS-C10 and application thereof, belonging to the technical field of microorganisms. The classification of Bacillus vallismortis YZS-C10 is named as Bacillus vallismortis (ZN)Bacillus vallismortis) YZS-C10, which has been deposited in China general microbiological culture Collection center on 9.7.2021, with the deposit numbers: CGMCC No.22857, with the preservation address of No. 3 Hospital No.1 Xilu of Beijing, Chaoyang. The bacillus vallismortis YZS-C10 has a good effect on the biodegradation of polyethylene plastics, provides new resources and solutions for the biodegradation of polyethylene wastes in the environment, and has great application prospects.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to bacillus vallismortis YZS-C10 and application thereof in biodegradation of polyethylene plastics.
Background
Plastics are important organic synthesesPolymerThe material has wide application. However, plastics are difficult to naturally degrade in the environment, and the white pollution caused by waste plastics is more and more serious. Polyethylene (PE) isEthyleneOne obtained by polymerizationThermoplastic resin. The polyethylene is odorless and nontoxic,the hand feeling is similar to wax, the low temperature resistance is excellent,chemical stabilityGood resistance to most acid and alkali. Is insoluble in normal temperatureSolvent(s)，Water absorptionThe size of the product is small, and the product is small,electrical insulationExcellent, and easy to accumulate in natural environment to cause secondary pollution. Therefore, safe disposal technology for waste plastics such as polyethylene is urgent, and currently, researches on biodegradation of waste plastics such as polyethylene are few, effective microbial resources are few, and treatment efficiency is low.
Mangrove forest grows in the intertidal zone of the coastal coast of tropical zone and subtropical zone and is submerged by periodic seawater for a long time. The method has the functions of flood prevention, wave prevention, storm prevention and bank protection, and has unique social and economic values in the aspects of maintaining the carbon-oxygen balance of the atmosphere, purifying the atmosphere and the water body environment and the like. The special habitat of mangrove determines the abundant microbial resources, and genetic resources different from other environmental microbial sources are stored.
Disclosure of Invention
The invention aims to provide a bacillus vallismortis strain and application thereof.
In order to achieve the purpose, the invention adopts the following technical scheme:
a strain of Bacillus vallismortis YZS-C10, which is classified and named as Bacillus vallismortis (Zb)Bacillus vallismortis) YZS-C10, which has been deposited in China general microbiological culture Collection center on 9.7.2021, with the deposit numbers: CGMCC No.22857, with the preservation address of No. 3 Hospital No.1 Xilu of Beijing, Chaoyang.
The colony characteristics and the thallus morphology of the bacillus vallismortis YZS-C10 are as follows:
culturing the YZS-C10 strain on an NA plate culture medium for 24 hours to form a colony which is light yellow, dull, round, free of mobility, gram-positive, spore-containing and capsular-free; the diameter of the colony is 3-4 mm.
Physiological and biochemical characteristics of the bacillus vallismortis YZS-C10:
the YZS-C10 strain is negative in catalase reaction, positive in V.P determination, negative in MR determination, positive in glucose acidogenesis test and glucose aerogenesis test, and negative in lemonNegative acid salt test, positive nitrate reduction, positive starch hydrolysis, negative indole test, negative malonic acid determination, and H production2Positive in S test.
The 16S rDNA sequence of the bacillus vallismortis YZS-C10 is compared with the sequence in the GenBank database, and the result shows that the YZS-C10 and the sequence in the GenBank database areBacillus vallismortisOn the same branch, the 16S rDNA sequence andBacillus vallismortis（KP994551.1) The similarity of the two types of the Chinese characters reaches 100 percent. Combining colony morphology, physiological and biochemical characteristics and 16S rDNA sequence analysis, and preliminarily identifying the bacillus vallismortis (A)Bacillus vallismortis）。
The invention has the advantages that:
the bacillus vallismortis YZS-C10 has good effect on the biodegradation of polyethylene plastics, provides new resources and solutions for the biodegradation of polyethylene wastes in the environment, and has great application prospect.
Drawings
FIG. 1 is a graph of the morphological features of PE films on an optical microscope after 30 days of degradation of Bacillus vallismortis YZS-C10. a, comparison; b, inoculating dead bacillus vallismortis YZS-C10.
FIG. 2 is a graph of the morphology of PE films in a scanning electron microscope after 30 days of degradation of Bacillus vallismortis YZS-C10. a, comparison; b, inoculating dead bacillus vallismortis YZS-C10.
FIG. 3 is a graph showing the change in water contact angle of PE films before and after degradation of Bacillus vallismortis YZS-C10.
FIG. 4 is a graph showing changes in functional groups on the surface of a PE film before degradation by Bacillus vallismortis YZS-C10.
FIG. 5 is a graph showing the change of functional groups on the surface of a PE film after degradation of Bacillus vallismortis YZS-C10.
Detailed Description
The present invention is further illustrated by the following examples.
A strain of Bacillus vallismortis YZS-C10, which is classified and named as Bacillus vallismortis (Zb)Bacillus vallismortis) YZS-C10, which has been deposited in China general microbiological culture Collection center on 9.7.2021, with the deposit numbers: CGMCC No.22857, storage AddressIs No. 3 Hospital No.1 Xilu, Beijing, Chaoyang, North Chen.
Example 1 isolation, screening and identification of Bacillus vallismortis
(1) Separation and screening processes:
319 isolates were isolated from the mangrove plant in Fujian by gradient dilution. Purifying the separated endophytic bacteria by a three-region scribing method, judging whether the bacterial strain is purified by microscopic examination, numbering the purified bacteria, picking a single bacterial colony, and transferring the single bacterial colony to an NA slant culture medium for storage and standby. Coating sterilized polyethylene powder 0.02 g/plate (plate diameter 9cm) on the surface of a carbon source-free mineral salt solid medium plate, carrying out streak culture on the obtained strain on the surface, screening single colonies which grow rapidly, and carrying out culture preservation on an NA plate medium. Through growth determination of a carbon source-free mineral salt culture medium with polyethylene as a unique carbon source, 7 strains with better polyethylene degradation effect are initially screened out, and finally, an endophytic bacterium with high efficiency in degrading polyethylene plastics is screened out and is marked as YZS-C10.
The culture medium for screening is a carbon source-free mineral salt culture medium and comprises the following components: 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，MnSO4·H20.001 g of O; the above components were dissolved in 1L of deionized water and the pH was adjusted to 7.0 with 1 mol/L NaOH. To the solid medium was added 1.5wt% agar.
Through the separation and screening work and multiple separation and purification, a rapidly-growing polyethylene-degraded endophytic bacterium Bacillus vallismortis YZS-C10 is obtained.
(2) Colony characteristics and colony morphology:
culturing the YZS-C10 strain on an NA plate for 24 hours to form a colony which is light yellow, matt, round, free of mobility, gram-positive, spore-containing and capsular; the diameter of the colony is 3-4 mm.
(3) Physiological and biochemical characteristics:
the YZS-C10 strain is negative in catalase reaction, positive in V.P determination, negative in MR determination, positive in glucose acid production test, negative in glucose gas production test, negative in citrate test, positive in nitrate reduction, positive in starch hydrolysis, negative in indole test, negative in malonic acid determination, and negative in H production2Positive in S test.
(4) 16S rDNA sequence analysis
The 16S rDNA gene sequence is shown in a nucleotide sequence table SEQ ID NO. 1. The measured 16S rDNA sequence is compared with the sequence in GenBank database, and the result shows that YZS-C10 and GenBank databaseBacillus vallismortisOn the same branch, the 16S rDNA sequence andBacillus vallismortis（KP994551.1) The similarity of the two types of the Chinese characters reaches 100 percent. Combining colony morphology, physiological and biochemical characteristics and 16S rDNA sequence analysis, and preliminarily identifying the bacillus vallismortis (A)Bacillus vallismortis）。
Example 2 degradation Effect of Bacillus vallismortis on polyethylene films
In order to evaluate the application of the bacillus vallismortis YZS-C10 in polyethylene degradation, a common polyethylene film is selected for degradation effect determination.
Dead Bacillus cereus (A. cereus)Bacillus vallismortis) YZS-C10 was activated on NA slant medium, and one loop was picked up and inoculated into NA medium (beef extract 3g, peptone 10g, NaCl 5g, agar 18g, water 1000ml, pH 7.0-7.2) for culture to logarithmic phase. Cutting polyethylene film into pieces of 1 × 1 cm2After weighing, the sample was sterilized with 75vol% ethanol for 2 h, and then the ethanol on the surface was evaporated in a clean bench with a sterile air flow. A conical flask was charged with 100 mL of a carbon source-free mineral salt liquid medium, 3 pieces of surface-sterilized polyethylene film were added, and the above-mentioned YZS-C10 culture solution in the logarithmic growth phase was inoculated in an inoculation amount of 1% (v/v). No inoculation treatment was used as a control, and 3 replicates were set for each treatment. Placing the conical flask in a constant-temperature shaking incubator for 30 d at 25 ℃ and 180 r/min, and sampling to determine the mass loss, surface appearance change, functional group change and hydrophobicity change of the polyethylene film.
Polyethylene film mass change determination: and (3) oscillating and washing the polyethylene membrane sheet for 2 h by using a 2wt% SDS solution, carrying out ultrasonic treatment for 15 min to remove a biological membrane on the surface, washing the membrane sheet for 3 times by using sterile water, placing the washed polyethylene membrane sheet in a dryer, drying for 48 h, and weighing. Mass loss rate of polyethylene (%) = (initial mass of polyethylene-mass after degradation)/initial mass × 100%.
And (3) determining the change of the microscopic morphology of the surface of the polyethylene film: washing the redundant culture medium on the surface with sterile water, fixing with 2.5wt% glutaraldehyde for 2 h, performing gradient dehydration with 30vol%, 50vol%, 70vol%, 90vol% and 100vol% ethanol for 15 min each time, and replacing with tert-butanol for 2 times for 30 min each time. Observing the surface appearance change of the processed sample by adopting an optical microscope; after the metal spraying is fixed, the micro-topography is observed under a scanning electron microscope (Hitachi SU 3800).
The change in the surface hydrophobicity of the polyethylene film was measured by a contact angle measuring instrument.
And (3) measuring the surface functional groups of the polyethylene film: after the cleaned polyethylene film is naturally dried, a Fourier infrared spectrometer is adopted for measurement, and the scanning wavelength range is 600-4000 cm-1Resolution of 4 cm-1And the number of scanning times is 32.
As can be seen from Table 1, when the polyethylene film was cultured in the above culture manner for 30 days, the polyethylene film mass after the YZS-C10 inoculation was reduced by 0.30mg on average, and the mass loss ratio was 5.85%. The control treatment was unchanged, indicating that polyethylene film sheets were degraded using YZS-C10.
TABLE 1 influence of YZS-C10 on the quality loss of polyethylene film
As can be seen from FIGS. 1 and 2, the inoculation of Bacillus vallismortis was observed under an optical microscope and a scanning electron microscope after 30 d of culture (Bacillus vallismortis) Compared with the control group, the YZS-C10 treated polyethylene film sheet has rough surface, disintegrated edges and obvious microbial erosion holes on the surface.
As can be seen from the view of figure 3,the result of the contact angle measuring instrument shows that the bacillus vallismortis is inoculatedBacillus vallismortis) The water contact angle of the YZS-C10-treated polyethylene film is 73.57 +/-2.17 degrees, the water contact angle of the contrast-treated polyethylene film is 91.40 +/-0.79 degrees, the water contact angle of the polyethylene film is reduced by 19.51 degrees which is 17.83 degrees lower than that of the contrast-treated polyethylene film, and therefore the water contact angle of the polyethylene film is obviously reduced by the bacterium inoculation treatment, and the polyethylene film is more easily attached and corroded by microorganisms.
As can be seen from FIGS. 4 and 5, Bacillus vallismortis (C.vallismortis)Bacillus vallismortis) YZS-C10 treated polyethylene film having an infrared spectrum of 1652 cm-1A new characteristic peak, the carbonyl peak, appeared. Carbonyl [ -C = O-bond]The occurrence of (A) is a basic sign of biodegradation of PE, indicating that the polyethylene is biodegraded.
The above description is only a preferred embodiment of the present invention, and all equivalent changes and modifications made in accordance with the claims of the present invention should be covered by the present invention.
SEQUENCE LISTING
<110> Minjiang academy
<120> Bacillus vallismortis YZS-C10 and application thereof
<130> 1
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 1424
<212> DNA
<213> SEQ ID NO.1
<400> 1
ttcggcggct ggctcctaaa ggttacctca ccgacttcgg gtgttacaaa ctctcgtggt 60
gtgacgggcg gtgtgtacaa ggcccgggaa cgtattcacc gcggcatgct gatccgcgat 120
tactagcgat tccagcttca cgcagtcgag ttgcagactg cgatccgaac tgagaacaga 180
tttgtgggat tggcttaacc tcgcggtttc gctgcccttt gttctgtcca ttgtagcacg 240
tgtgtagccc aggtcataag gggcatgatg atttgacgtc atccccacct tcctccggtt 300
tgtcaccggc agtcacctta gagtgcccaa ctgaatgctg gcaactaaga tcaagggttg 360
cgctcgttgc gggacttaac ccaacatctc acgacacgag ctgacgacaa ccatgcacca 420
cctgtcactc tgcccccgaa ggggacgtcc tatctctagg attgtcagag gatgtcaaga 480
cctggtaagg ttcttcgcgt tgcttcgaat taaaccacat gctccaccgc ttgtgcgggc 540
ccccgtcaat tcctttgagt ttcagtcttg cgaccgtact ccccaggcgg agtgcttaat 600
gcgttagctg cagcactaag gggcggaaac cccctaacac ttagcactca tcgtttacgg 660
cgtggactac cagggtatct aatcctgttc gctccccacg ctttcgctcc tcagcgtcag 720
ttacagacca gagagtcgcc ttcgccactg gtgttcctcc acatctctac gcatttcacc 780
gctacacgtg gaattccact ctcctcttct gcactcaagt tccccagttt ccaatgaccc 840
tccccggttg agccgggggc tttcacatca gacttaagaa accgcctgcg agccctttac 900
gcccaataat tccggacaac gcttgccacc tacgtattac cgcggctgct ggcacgtagt 960
tagccgtggc tttctggtta ggtaccgtca aggtgccgcc ctatttgaac ggcacttgtt 1020
cttccctaac aacagagctt tacgatccga aaaccttcat cactcacgcg gcgttgctcc 1080
gtcagacttt cgtccattgc ggaagattcc ctactgctgc ctcccgtagg agtctgggcc 1140
gtgtctcagt cccagtgtgg ccgatcaccc tctcaggtcg gctacgcatc gtcgccttgg 1200
tgagccgtta cctcaccaac tagctaatgc gccgcgggtc catctgtaag tggtagccga 1260
agccaccttt tatgtctgaa ccatgcggtt caaacaacca tccggtatta gccccggttt 1320
cccggagtta tcccagtctt acaggcaggt tacccacgtg ttactcaccc gtccgccgct 1380
aacatcaggg agcaagctcc catctgtccg ctcgacttgc atgt 1424
Claims (2)
1. A Bacillus vallismortis YZS-C10 is characterized in that: the classification of the Bacillus vallismortis YZS-C10 is named as Bacillus vallismortis (Bacillus vallismortis) YZS-C10, which has been deposited in China general microbiological culture Collection center on 9.7.2021, with the deposit numbers: CGMCC No.22857, with the preservation address of No. 3 Hospital No.1 Xilu of Beijing, Chaoyang.
2. Use of the bacillus vallismortis YZS-C10 of claim 1 in the biodegradation of polyethylene plastics.
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| CN202111151301.0A CN113801822B (en) | 2021-09-29 | 2021-09-29 | Bacillus cereus YZS-C10 and application thereof |
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| CN202111151301.0A CN113801822B (en) | 2021-09-29 | 2021-09-29 | Bacillus cereus YZS-C10 and application thereof |
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| Publication Number | Publication Date |
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| CN113801822A true CN113801822A (en) | 2021-12-17 |
| CN113801822B CN113801822B (en) | 2024-04-12 |
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| CN115960782A (en) * | 2022-12-22 | 2023-04-14 | 中国科学院南海海洋研究所 | Marine polyethylene plastic degrading bacterium and application thereof |
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| 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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