CN113801822B - Bacillus cereus YZS-C10 and application thereof - Google Patents

Bacillus cereus YZS-C10 and application thereof Download PDF

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CN113801822B
CN113801822B CN202111151301.0A CN202111151301A CN113801822B CN 113801822 B CN113801822 B CN 113801822B CN 202111151301 A CN202111151301 A CN 202111151301A CN 113801822 B CN113801822 B CN 113801822B
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yzs
bacillus cereus
polyethylene
bacillus
biodegradation
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CN113801822A (en
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袁宗胜
刘芳
谢宝贵
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Minjiang University
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    • BPERFORMING OPERATIONS; TRANSPORTING
    • B29WORKING OF PLASTICS; WORKING OF SUBSTANCES IN A PLASTIC STATE IN GENERAL
    • B29BPREPARATION OR PRETREATMENT OF THE MATERIAL TO BE SHAPED; MAKING GRANULES OR PREFORMS; RECOVERY OF PLASTICS OR OTHER CONSTITUENTS OF WASTE MATERIAL CONTAINING PLASTICS
    • B29B17/00Recovery of plastics or other constituents of waste material containing plastics
    • YGENERAL 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
    • Y02TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02WCLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
    • Y02W30/00Technologies for solid waste management
    • Y02W30/50Reuse, recycling or recovery technologies
    • Y02W30/62Plastics recycling; Rubber recycling

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  • Environmental & Geological Engineering (AREA)
  • Mechanical Engineering (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

The invention provides bacillus cereus YZS-C10 and application thereof, and belongs to the technical field of microorganisms. The classification of the bacillus cereus YZS-C10 is named as bacillus cereus @Bacillus vallismortis) YZS-C10 was deposited in China general microbiological culture Collection center, with the accession number: CGMCC No.22857, the preservation address is Hospital No.1 and No. 3 of North Chen West road in the Chaoyang area of Beijing city. The bacillus cereus 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.

Description

Bacillus cereus YZS-C10 and application thereof
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to bacillus cereus YZS-C10 and application thereof in biodegradation of polyethylene plastics.
Background
Plastics are important organic synthesesPolymerThe material is very widely applied. However, plastics are difficult to degrade naturally in the environment, and the "white pollution" caused by waste plastics is also more and more serious. Polyethylene (PE) isEthyleneOne obtained by polymerizationThermoplastic resin. Polyethylene is odorless, nontoxic, wax-like in hand feeling, has excellent low temperature resistance,chemical stabilityAnd the product is good in resistance to most of acid and alkali. Is insoluble in general at normal temperatureSolvent(s)Water absorptionThe size of the product is small and the product is easy to be manufactured,electrical insulation propertyExcellent, is easy to accumulate in natural environment, and causes secondary pollution. Therefore, the technology of disposing of plastic such as waste polyethylene is urgent, and the current research on biodegradation of plastic waste such as polyethylene is less and effectiveLittle microbial resource and low treatment efficiency.
Mangrove grows in the tropical, subtropical offshore intertidal zone and is immersed in periodic seawater for a long period of time. The water-saving type flood control system has the functions of flood prevention, wave prevention, storm prevention, dike protection and the like, 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 environment and the like. The special habitat of mangrove determines its abundant microbial resources, and reserves genetic resources different from other environmental microbial sources.
Disclosure of Invention
The invention aims to provide a bacillus cereus strain and application thereof.
In order to achieve the above purpose, the invention adopts the following technical scheme:
bacillus cereus YZS-C10, which is classified and named as Bacillus cereus @Bacillus vallismortis) YZS-C10 was deposited in China general microbiological culture Collection center, with the accession number: CGMCC No.22857, the preservation address is Hospital No.1 and No. 3 of North Chen West road in the Chaoyang area of Beijing city.
The colony characteristics and the thallus morphology of the dead bacillus cereus YZS-C10 are as follows:
the bacterial colony formed by culturing YZS-C10 strain on NA flat plate culture medium for 24h is light yellow, matt, round, free of fluidity, gram positive, spore and capsule-free; the colony diameter is 3-4mm.
Physiological and biochemical characteristics of the bacillus cereus YZS-C10:
the YZS-C10 strain has negative contact enzyme reaction, V.P measurement is positive, MR measurement is negative, glucose acidogenesis test is positive, glucose acidogenesis test is negative, citrate test is negative, nitrate reduction is positive, starch hydrolysis is positive, indole test is negative, malonic acid measurement is negative, and H is produced 2 S test positive.
The result of comparing the 16S rDNA sequence of the dead bacillus cereus YZS-C10 with the sequences in the GenBank database shows that the YZS-C10 is compared with the sequences in the GenBank databaseBacillus vallismortisOn one branch, its 16S rDNA sequence andBacillus vallismortisKP994551.1) The similarity of (2) reaches 100%. Combining colony morphology, physiological and biochemical characteristics and 16S rDNA sequence analysis, and primarily identifying the bacillus cereus as the bacillus cereus @Bacillus vallismortis)。
The invention has the advantages that:
the bacillus cereus 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
Figure 1 topographical features of PE films in an optical microscope after 30 days of degradation by Bacillus cereus YZS-C10. a control; b inoculating bacillus cereus YZS-C10.
Figure 2 is a graph showing the morphological characteristics of PE films in a scanning electron microscope after 30 days of degradation of Bacillus cereus YZS-C10. a control; b inoculating bacillus cereus YZS-C10.
FIG. 3 shows the change of water contact angle of PE film before and after degradation of Bacillus cereus YZS-C10.
FIG. 4 shows the change of functional groups on the surface of PE film before degradation of Bacillus cereus YZS-C10.
FIG. 5 shows the change of functional groups on the surface of PE film after degradation of Bacillus cereus YZS-C10.
Detailed Description
The invention is further illustrated by the following examples.
Bacillus cereus YZS-C10, which is classified and named as Bacillus cereus @Bacillus vallismortis) YZS-C10 was deposited in China general microbiological culture Collection center, with the accession number: CGMCC No.22857, the preservation address is Hospital No.1 and No. 3 of North Chen West road in the Chaoyang area of Beijing city.
EXAMPLE 1 isolation screening and identification of Bacillus cereus
(1) And (3) separating and screening:
319 isolates were isolated from Fujian mangrove plants by gradient dilution. Purifying the separated endophytic bacteria by a three-zone streak method, judging whether the bacterial strain is purified by microscopic examination, numbering the purified bacteria, picking single bacterial colonies, and transferring the single bacterial colonies to an NA slant culture medium for storage. The surface of a plate of a carbon source-free mineral salt solid medium is coated with 0.02 g/plate (the diameter of the plate is 9 cm) of sterilized polyethylene powder, the strain obtained above is subjected to streak culture on the surface, and single colonies which grow rapidly are screened to be cultured and stored on an NA plate medium. And 7 strains with better polyethylene degradation effect are initially screened out through growth measurement of a carbon source-free mineral salt culture medium with polyethylene as a unique carbon source, and endophytic bacteria with high-efficiency degradation polyethylene plastic are finally screened out, and marked as YZS-C10.
The culture medium for screening is a mineral salt culture medium without carbon source, and comprises the following components: k (K) 2 HPO 4 ·3H 2 O 0.7 g,KH 2 PO 4 0.7 g,NH 4 NO 3 1.0 g,NaCl 0.5 g,MgSO 4 ·7H 2 O 0.5 g,FeSO 4 ·7H 2 O 0.002 g,ZnSO 4 ·7H 2 O 0.002 g,MnSO 4 ·H 2 O0.001 g; the above components were dissolved in 1L deionized water and the pH was adjusted to 7.0 with 1 mol/L NaOH. 1.5wt% agar was added to the solid medium.
Through the separation and screening work, the bacillus cereus YZS-C10 which is the endophyte of polyethylene degradation and grows rapidly is obtained through multiple separation and purification.
(2) Colony characteristics and colony morphology:
after the YZS-C10 strain is cultured on an NA plate for 24 hours, a colony is light yellow, matt, round, free of fluidity, gram-positive, spore-bearing and capsule-free; the colony diameter is 3-4mm.
(3) Physiological and biochemical characteristics:
the YZS-C10 strain has negative contact enzyme reaction, V.P measurement is positive, MR measurement is negative, glucose acidogenesis test is positive, glucose acidogenesis test is negative, citrate test is negative, nitrate reduction is positive, starch hydrolysis is positive, indole test is negative, malonic acid measurement is negative, and H is produced 2 S test positive.
(4) 16S rDNA sequence analysis
The 16S rDNA gene sequence is shown in a nucleotide sequence table SEQ ID NO. 1. The result of comparing the detected 16S rDNA sequence with the sequences in GenBank database shows that YZS-C10 is compared withBacillus vallismortisOn one branch, its 16S rDNA sequence andBacillus vallismortisKP994551.1) The similarity of (2) reaches 100%. Combining colony morphology, physiological and biochemical characteristics and 16S rDNA sequence analysis, and primarily identifying to be bacillus cereus @Bacillus vallismortis)。
EXAMPLE 2 degradation Effect of Bacillus cereus on polyethylene film
In order to evaluate the application of the bacillus cereus YZS-C10 in polyethylene degradation, a common polyethylene film is selected for degradation effect measurement.
Bacillus cereusBacillus vallismortis) YZS-C10 is activated on NA slant culture medium, and one loop is selected and inoculated into NA culture medium (beef extract 3g, peptone 10g, naCl 5g, agar 18g, water 1000ml, pH 7.0-7.2) for culturing to logarithmic phase. Cutting polyethylene film into 1×1 cm pieces 2 After weighing, 2 h was sterilized with 75vol% ethanol, and then the surface ethanol was volatilized off with a sterile air stream in an ultra clean bench. 100 mL carbon-free mineral salt liquid culture medium is added into a conical flask, 3 pieces of surface sterilized polyethylene film are added, and the YZS-C10 logarithmic growth phase culture solution is inoculated with an inoculum size of 1% (v/v). The control group was a sterile treatment, and 3 replicates were set for each treatment. After placing the conical flask in a constant temperature shaking incubator at 25 ℃ and 180 r/min for culturing for 30 d, samples are taken to measure the mass loss, the surface morphology change, the functional group change and the hydrophobicity change of the polyethylene film.
And (3) measuring the mass change of the polyethylene film: the polyethylene film sheet 2 h was washed with 2wt% SDS solution by shaking, sonicated for 15 min, the biofilm on the surface was removed, the film sheet was washed 3 times with sterile water, and the washed polyethylene film sheet was dried in a desiccator for 48 h and then weighed. Mass loss rate (%) = (initial mass of polyethylene-mass after degradation)/initial mass×100% of polyethylene.
And (3) measuring the microscopic morphology change of the surface of the polyethylene film: excess medium on the surface was rinsed with sterile water, fixed with 2.5wt% glutaraldehyde for 2 h, and dehydrated sequentially with 30vol%, 50vol%, 70vol%, 90vol%, 100vol% ethanol for 15 min each, and replaced with t-butanol for 2 times each for 30 min. Observing the surface morphology change of the treated sample by adopting an optical microscope; microscopic morphological features were observed under a scanning electron microscope (Hitachi SU 3800) after fixed metal spraying.
The change in surface hydrophobicity of the polyethylene film was measured using a contact angle measuring instrument.
Surface functional group measurement of polyethylene film: naturally drying the cleaned polyethylene film, measuring with Fourier infrared spectrometer, and scanning wavelength range of 600-4000 cm -1 Resolution 4 cm -1 The number of scans was 32.
As can be seen from Table 1, when the polyethylene film was cultured to 30 d in the above-described culture manner, the mass of the polyethylene film inoculated with YZS-C10 treatment was reduced by 0.30mg on average, and the mass loss ratio was 5.85%. The control treatment was unchanged, indicating degradation of the polyethylene film by YZS-C10.
TABLE 1 influence of YZS-C10 on the mass loss of polyethylene films
As can be seen from FIGS. 1 and 2, after 30 d culture, the inoculated Bacillus cereus was observed under an optical microscope and a scanning electron microscopeBacillus vallismortis) Compared with the control group, the YZS-C10 treated polyethylene film has rough surface, disintegrates at the edge, and has obvious microorganism erosion holes on the surface.
As can be seen from FIG. 3, the results of the contact angle measurement show that the bacillus cereus is inoculatedBacillus vallismortis) The water contact angle of the polyethylene film treated by YZS-C10 is 73.57 +/-2.17 degrees, the water contact angle of the polyethylene film treated by the control treatment is 91.40+/-0.79 degrees, which is lower than that of the polyethylene film treated by the control treatment by 17.83 degrees, and the water contact angle is reduced by 19.51 percent, which shows that the water contact angle of the polyethylene film treated by the sterilization treatment is obviously reduced, and the polyethylene film is more easily adhered and corroded by microorganisms.
As can be seen from FIGS. 4 and 5, the inoculation of Bacillus cereusBacillus vallismortis) The infrared spectrum of the polyethylene film treated by YZS-C10 is 1652 cm -1 A new characteristic peak, i.e. a carbonyl peak, appears. Carbonyl [ -C=O-bond]The appearance of (2) is a fundamental sign of PE biodegradation, indicating that polyethylene is biodegradable.
The foregoing description is only of the preferred embodiments of the invention, and all changes and modifications that come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.
SEQUENCE LISTING
<110> Minjiang college
<120> Bacillus vallismortis YZS-C10 strain 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. The bacillus cereus YZS-C10 strain is characterized in that: the classification of the dead bacillus cereus YZS-C10 is named as dead bacillus cereus @Bacillus vallismortis) YZS-C10 is preserved in China general microbiological culture Collection center (CGMCC) at 7 and 9 days of 2021, with a preservation number of CGMCC No.22857 and a preservation address of North Chenxi Lu No.1 and 3 in the Chaoyang area of Beijing; the 16S rDNA gene sequence of the dead bacillus cereus YZS-C10 is shown in SEQ ID NO. 1.
2. The application of the dead bacillus cereus YZS-C10 in the biodegradation of polyethylene plastics, which is characterized in that: the temperature of the degradation was 25 ℃.
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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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JP2004166542A (en) * 2002-11-18 2004-06-17 Japan Science & Technology Agency New plastic decomposing bacterium
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