CN116640699B - Streptomyces viridochromogenes and application thereof in degradation of natural rubber - Google Patents
Streptomyces viridochromogenes and application thereof in degradation of natural rubber Download PDFInfo
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
- B09B—DISPOSAL OF SOLID WASTE
- B09B3/00—Destroying solid waste or transforming solid waste into something useful or harmless
- B09B3/60—Biochemical treatment, e.g. by using enzymes
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
- B09B—DISPOSAL OF SOLID WASTE
- B09B2101/00—Type of solid waste
- B09B2101/80—Rubber waste, e.g. scrap tyres
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/465—Streptomyces
-
- 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
Abstract
The invention discloses a Streptomyces viridochromogenes FZUW-1 and application thereof in degrading natural rubber. The strain is classified and named as streptomyces viridochromogenesStreptomyes viridochromogenes) The strain is preserved in China general microbiological culture Collection center (CGMCC) at 28 days of 3 months of 2023, and the preservation number is CGMCC No.26909. The streptomyces viridochromogenes FZUW-1 can grow on the surface of the rubber by taking the natural rubber as a carbon source, so that the carbon chain of the natural rubber is oxidized, the carbon long chain of the natural rubber is damaged, and the purpose of degrading the natural rubber is achieved. The invention provides strain resources and a new way for degrading natural rubber by microorganisms.
Description
Technical Field
The invention belongs to the technical field of microorganism application, and particularly relates to streptomyces viridochromogenes and application thereof in degrading natural rubber.
Background
Natural rubber (Naturalrubber) is a natural polymer compound with cis-1, 4-polyisoprene as a main component, and the proportion of cis-1, 4-polyisoprene in the natural rubber reaches 91% -94%. At present, the natural rubber waste is also obviously increased, the waste rubber is one of solid waste, and the source of the waste rubber is mainly waste rubber products, namely discarded tires, manual tires, rubber tubes, rubber belts, industrial sundries and the like, are accumulated after being discarded, and in addition, a part of waste materials and waste products are generated in the production process of rubber product factories. Because the rubber is difficult to decompose naturally, the rubber occupies land for a long time, and is more fatal, serious in soil environmental pollution is caused, and a large amount of toxic gas generated by incineration also has a certain harm to the atmospheric environment.
The degradation method of rubber is also widely used at present, and comprises various methods such as thermomechanical degradation, microwave degradation, ultrasonic degradation, redox degradation, microbial degradation and the like. These traditional physicochemical degradation methods suffer from a number of disadvantages: high heat consumption, low economic benefit, etc.
Biological methods have been considered as methods with low energy consumption and good environmental benefits, and the existing researchers can degrade rubber by using lactobacillus plantarum, and the mass loss reaches 4% after two weeks of degradation, so that the carbon content of the rubber is reduced. Bacillus cereus is also used as a rubber degradation strain by researchers, conventional vulcanized and high-efficiency vulcanized rubber is subjected to experiments by using bacillus, and the weakening of a C peak of the rubber is judged by XPS, so that the reduction of the quality of the rubber under degradation is proved. The method is characterized in that a plurality of microorganisms for degrading rubber are separated from soil, the mass loss analysis of the rubber after degradation of various single bacteria finds that the mass loss of various single bacteria is below 10%, and the research considers that the synergistic effect of various bacteria plays an important role. Actinomycota is considered to be a major key to the management of rubber problems, and nocardia and streptomyces in actinomycota are considered to have excellent degradation potential.
In summary, the large amount of rubber waste has become a very troublesome problem, and the disposal of "black waste" is not slow as is faced by degradation of plastics. Numerous studies have shown that rubber is degradable, and that physicochemical methods such as thermo-mechanical and microwave have matured, but with little energy consumption and economic benefit. Thus, the development of methods utilizing microbial treatment is vigorous, and research on microbial degradation of rubber has been focused, so that the exploration of new microorganisms for degrading rubber is of great importance.
Disclosure of Invention
In view of the above, the present invention aims to provide a Streptomyces viridochromogenes and its application in degradation of natural rubber.
In order to achieve the above purpose, the invention adopts the following technical scheme:
the invention firstly provides a strain of streptomyces viridochromogenes FZUW-1, which has been deposited in China general microbiological culture Collection center (China general microbiological culture Collection center) for 3-month 28 of 2023, with the deposit number: CGMCC No.26909, the preservation address is: no.1 and No. 3 of the north cinquefoil of the morning sun area of beijing city.
Streptomyces viridochromogenes-FZUW-1 is a gram-positive bacterium; the colony in ISP2 solid culture medium is white round in earlier stage, and the colony is transformed from white round to dark green round after being cultured at 37 ℃ until the third day.
The invention also provides application of the streptomyces viridochromogenes FZUW-1 in degrading natural rubber.
The invention also provides a method for degrading natural rubber by using streptomyces viridochromogenes FZUW-1, which comprises the following steps: and (3) performing amplification culture on streptomyces viridochromogenes (FZUW-1), co-culturing with natural rubber, and finally drying the reactant to obtain degradation products.
Further, the method for degrading the natural rubber by using streptomyces viridochromogenes FZUW-1 comprises the following steps:
(1) Activating, purifying and amplifying culturing the streptomyces viridochromogenes FZUW-1;
(2) Co-culturing the bacterial liquid obtained in the step (1) with natural rubber;
(3) And (3) drying the reactant obtained in the step (2) to obtain degradation products.
Further, the method for degrading the natural rubber by using the streptomyces viridochromogenes FZUW-1 comprises the following steps:
(1) Activating and purifying streptomyces viridochromogenes FZUW-1 and preparing cell suspension;
(2) Co-culturing the cell suspension obtained in the step (1) with non-vulcanized rubber or vulcanized rubber;
(3) And (3) obtaining the degraded rubber product in the step (2).
From the scheme, the invention has the following beneficial effects:
the streptomyces viridochromogenes FZUW-1 can grow on a rubber culture medium, has a certain degradation effect on non-vulcanized natural rubber and vulcanized rubber, can be used for treating waste rubber, and provides strain resources and technical support for microbial degradation of rubber.
Drawings
FIG. 1 is a morphology of Streptomyces viridochromogenes FZUW-1. a, a morphology diagram of colony in rubber culture medium; b, morphology of colony in ISP2 solid culture medium; c, morphological image under microscope.
FIG. 2 is a phylogenetic tree of Streptomyces viridochromogenes FZUW-1.
FIG. 3 is a FT-IR diagram of a degraded unvulcanized rubber sheet of Streptomyces viridochromogenes FZUW-1.
FIG. 4 is a graph showing the growth of Streptomyces viridochromogenes FZUW-1 on the surface of unvulcanized rubber and an SEM graph.
FIG. 5 is a graph showing the mass loss and gel degree of unvulcanized rubber with Streptomyces viridochromogenes FZUW-1 over a four week period.
FIG. 6 is an SEM image of a degraded vulcanized rubber sheet of Streptomyces viridochromogenes FZUW-1.
Detailed Description
In order to make the contents of the present invention more easily understood, the technical scheme of the present invention will be further described with reference to the specific embodiments, but the present invention is not limited thereto.
In the invention, the formula of the MSM solid culture medium is as follows: naCl0.1g, ca (NO) 3 ) 2 0.2g、NH 4 NO 3 2.0g、MgSO 4 ·7H 2 O0.2g、CaCl20.02g、FeSO 4 ·7H 2 O0.001g、K 2 HPO 4 0.8g、KH 2 PO 4 1g、H 2 O1L, 20g of agar powder; ph=7.
In the invention, the fresh natural rubber latex is purchased from three trees international control stock limited company in thailand, and the dry rubber content is 60.2%. The preparation method of the concentrated natural rubber latex comprises the following steps: adding fresh natural rubber latex into 0.002% tween-20 solution, controlling the mass ratio of fresh natural rubber latex to tween-20 to be 1: centrifuging at 10000rpm for 10min, discarding precipitate, and collecting supernatant to obtain concentrated natural rubber latex.
In the invention, the ISP2 liquid culture medium comprises the following components: 4.0g of yeast extract, 10.0g of malt extract, 4.0g of glucose and H 2 O1L。
In the invention, the ISP2 solid culture medium comprises the following components: 4.0g of yeast extract, 10.0g of malt extract, 4.0g of glucose and H 2 O1L, 20g of agar powder.
In the invention, the preparation method of the unvulcanized rubber sheet comprises the following steps: fresh natural rubber latex is stirred at 600rpm for 60min, excessive bubbles are removed under ultrasonic state by using T-118 high-efficiency non-silicon defoamer (purchased from Jiangsu Teng auxiliary agent Co., ltd.), the mixture is poured into a mould, the thickness of the rubber sheet is controlled to be 2+/-0.2 mm, the rubber sheet is dried at 60 ℃ for 24h, and the rubber sheet is cut into sheets of 2cm multiplied by 2cm after drying and molding.
In the invention, the vulcanized rubber sheet is purchased from Yangzi medical instruments Inc. of Haimen, jiangsu province.
EXAMPLE 1 isolation of FZUW-1 Strain
Adding 1g of garbage pile soil into 10mL of sterile water, and carrying out gradient dilution to 10 -3 ～10 -4 . 100. Mu.L of the diluted solution was applied to MSM solid medium, and then sterilized concentrated natural rubber latex was added to the mixture to cover the coated MSM solid medium, and the mixture was cultured in an incubator at 37℃for 5 days. Single colonies were picked and purified 5 times in ISP2 liquid medium to obtain a strain capable of growing on a medium with natural rubber as a carbon source, which was designated FZUW-1.
EXAMPLE 2 identification of FZUW-1 Strain
Morphological identification (fig. 1): the colony of FZUW-1 strain on ISP2 solid culture medium is white round in early stage, white colony starts to turn to dark green gradually after culturing for about 72h, and the colony surface is filiform and spread outwards when the colony is adhered tightly to the culture medium and goes deep into the culture medium. When observed with an electron microscope, the bacteria were found to have a long oval chain structure.
Identification by 16SrDNA microorganism identification System: the 16SrDNA universal primer is adopted to carry out the PCR amplification of the 16SrDNA gene by taking the FZUW-1 strain genome DNA as a template, and the primers are as follows:
27F：5'-AGTTTGATCMTGGCTCAG-3'，
1492R：5'-GGTTACCTTGTTACGACTT-3'。
the PCR amplified product was subjected to electrophoresis on 1% agarose, then cut into gel, and recovered for sequencing (SEQ ID NO. 1). As a result of sequence alignment on NCBI, it was found that FZUW-1 strain has a similarity of up to 98% with Streptomyces viridochromogenes. Construction of developmental tree the developmental tree was constructed using the method of neighbor joining in MEGA7.0 software as shown in fig. 2.
EXAMPLE 3 degradation of non-vulcanized film by FZUW-1 Strain
(1) The purified FZUW-1 strain was inoculated into 30mL of sterilized ISP2 liquid medium and cultured in a shaker at 120rpm and 37℃for 3d. Pouring the culture solution into a sterilized centrifuge tube, centrifuging at 8000rpm for 10min, collecting precipitate, cleaning with physiological saline for three times, and re-suspending to obtain FZUW-1 seed solution with concentration of 3.6X10 9 CFU/mL。
(2) Adding 0.5g of unvulcanized rubber sheet into a 50mLMSM liquid culture medium containing 0.002% Tween-20, sterilizing at 121 ℃ for 20min to obtain a rubber culture medium, shaking up FZUW-1 seed liquid, and then mixing according to the seed liquid: medium = 1:5v/v ratio was inoculated thereto and placed in a shaking table at 130rpm,37℃for 30d. Equivalent amounts of MSM liquid culture were used as controls instead of FZUW-1 seed liquid.
(3) And (3) after the culture in the step (2) is finished, taking out the unvulcanized rubber sheet, and measuring the degradation condition of the unvulcanized rubber sheet after vacuum drying at 60 ℃ for 3d.
As shown in FIG. 3, after 30 days of degradation, the change of the rubber is mainly found to have a plurality of functional groups, and in summary, FZUW-1 is mainly used for breaking carbon-carbon double bonds to generate a large number of oxygen-containing functional groups, and a part of methyl groups and methylene groups are consumed in the oxidation process, so that the purpose of degrading the rubber is achieved by the mechanism.
As shown in FIG. 4a, FZUW-1 was able to grow on the surface of unvulcanized rubber with the rubber as a carbon source. FIG. 4b shows the adhesion of FZUW-1 to the rubber surface under a scanning electron microscope, which shows that a large number of Streptomyces species with long chain structure grow on the rubber surface.
As shown in FIG. 5, FZUW-1 can reduce the mass of unvulcanized rubber and cause more soluble substances to appear on the rubber surface, resulting in a decrease in the gelation degree of unvulcanized rubber.
EXAMPLE 4 degradation of vulcanized rubber sheet by FZUW-1 Strain
(1) The purified FZUW-1 strain was inoculated into 30mL of sterilized ISP2 liquid medium and cultured in a shaker at 120rpm and 37℃for 3d. Pouring the culture solution into a sterilized centrifuge tube, centrifuging at 8000rpm for 10min, collecting precipitate, cleaning with physiological saline for three times, and re-suspending to obtain FZUW-1 seed solution with concentration of 3.6X10 9 CFU/mL。
(2) Taking 0.5g vulcanized rubber sheet (cutting the sheet into sheet shape of 2cm×2cm, adding absolute ethanol, soaking for 24 hr to remove harmful substances in vulcanized rubber, naturally air-drying), adding into 50 mM SM liquid culture medium containing 0.002% Tween 20, and sterilizing at 121deg.C for 20 min. Shaking the seed liquid obtained in the step (1) uniformly, and then pressing the seed liquid: medium=1:5 v/v was added to the sterilized medium, and the mixture was put into a shaker and degraded at 130rpm and 37 ℃. Each set of degradation experiments was repeated three times. Equivalent amounts of MSM liquid culture were used as controls instead of FZUW-1 seed liquid. Degradation times were set to 7d, 14d, 21d, 28d.
(3) Taking out the vulcanized rubber sheet after degradation in the step (2), and measuring degradation condition after drying for 3d under vacuum condition at 60 ℃.
As shown in fig. 6, the rubber surface after degradation 30d is shown in fig. 6 (d) compared with the rubber surface before degradation, and the surface after degradation shows a large number of corrosion pits, the surface of the whole rubber is rugged, a large number of cracks are carried in the corrosion pits, and the surface shows a cracking form with the center outwards.
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.
Claims (3)
1. Streptomyces viridochromogenes FZUW-1, which is characterized in that: the classification of the strain is named as Streptomyces viridochromogenesStreptomyes viridochromogenes) The microbial strain has been deposited in China general microbiological culture Collection center, with a deposit address of: the preservation number of the Beijing city Chaoyang area North Chen Xili No.1 and 3 is: CGMCC No.26909.
2. The use of streptomyces viridochromogenes FZUW-1 according to claim 1 for degrading natural rubber.
3. A method for degrading natural rubber by using the streptomyces viridochromogenes FZUW-1 as claimed in claim 1, characterized in that: and (3) preparing seed liquid by performing amplification culture on the streptomyces viridochromogenes FZUW-1, and co-culturing the streptomyces viridochromogenes FZUW-1 seed liquid with natural rubber.
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| CN103131656A (en) * | 2013-03-06 | 2013-06-05 | 江苏省农业科学院 | Streptomyces YT027 and application thereof |
| CN106434475A (en) * | 2016-11-01 | 2017-02-22 | 滕州市悟通香料有限责任公司 | Streptomyces polysaccharide degradation bacterium as well as culture method and application thereof |
| WO2021000637A1 (en) * | 2019-07-01 | 2021-01-07 | 浙江大学 | New daptomycin-producing streptomyces strain and application thereof |
| CN112522130A (en) * | 2020-09-30 | 2021-03-19 | 仲恺农业工程学院 | Streptomyces for preventing and treating southern blight of rubber banyan, microbial inoculum thereof, and preparation method and application thereof |
| CN115927057A (en) * | 2021-11-04 | 2023-04-07 | 北京工商大学 | Streptomyces strain YP1 and application thereof |
| CN114292777A (en) * | 2021-12-22 | 2022-04-08 | 中国科学院东北地理与农业生态研究所 | Cellulose-degrading bacterium and application thereof |
| CN114317361A (en) * | 2021-12-31 | 2022-04-12 | 北方民族大学 | Streptomyces new strain and separation method and application thereof |
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