CN112063567A - Rhodococcus pyridinivorans and application thereof in production of PHBV - Google Patents
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Abstract
The invention discloses Rhodococcus pyridinivorans and application thereof in production of PHBV. Rhodococcus pyridinivorans (Rhodococcus pyridinivorans) P23, deposited at the China Center for Type Culture Collection (CCTCC) on 7/8/2019, addresses: China-Wuhan university, the preservation number is: CCTCC NO: m2019609. The bacterial strain Rhodococcus pyridinivorans P23 can efficiently synthesize the copolymer PHBV of 3-hydroxybutyric acid and 3-hydroxyvaleric acid by using terephthalic acid or terephthalate as a substrate in a mineral salt culture medium. The PHBV synthesized by the bacterial strain Rhodococcus pyridinivorans P23 has a monomer content of 3-hydroxyvaleric acid of more than 60 percent, a weight average molecular weight of higher than 600kDa, excellent physical properties and thermodynamic properties and good application prospect.
Description
Technical Field
The invention relates to the technical field of degradable plastics, in particular to rhodococcus pyridinivorans and application thereof in production of PHBV.
Background
Plastics are widely produced and used because of their advantages of being lightweight, strong, easy to process, inexpensive, and a good insulator. According to the statistics of 'plastics Europe', 3.48 million tons of plastics are produced worldwide in 2017, while China is the largest plastic producing country in the world, and the produced plastics account for 29.4% of the world (https:// www.plasticseurope.org/en). The application of the plastic brings great convenience to the life of people, and serious energy and resource waste and environmental pollution are caused due to excessive use, improper recovery and treatment and the like. As early as 2007 in 12 months, china issued "plastic restriction order", which clearly stipulates: "from 2008, 6/1, production and sale are prohibited nationwide, and plastic shopping bags with a thickness of less than 0.025 mm are used". This so-called old version of the "plastic limit order" has been formally executed for 12 years since 6/1 of 2008. With the lapse of time, its drawbacks are increasingly highlighted. In recent years, the economic rise of the internet has led to a great deal of plastic consumption from a number of new growth states. Therefore, the recent national development and improvement committee and the outside of ecological environment issue "opinions on further enhancing plastics pollution control". By the end of 2020, the use of non-degradable disposable plastic straws is forbidden in catering industry nationwide; the service of dining hall food in built-up district and scenic spot of city above grade is forbidden to use non-degradable disposable plastic tableware. By the end of 2022, dining hall food service in built-up districts and scenic spots in county cities, the use of non-degradable disposable plastic tableware is forbidden. By 2025, the consumption intensity of the non-degradable disposable plastic tableware in the field of urban catering takeouts above grade is reduced by 30%.
The biodegradable plastic can be used for overcoming the defect that the traditional petroleum-based plastic is not degradable or difficult to degrade. Polyhydroxyalkanoate (PHA) is an intracellular polyester synthesized by microorganisms, and has the characteristics of degradability, biocompatibility and the like. The PHA is divided into short chain PHA (C3-C5) and medium and long chain PHA (C6-C14) according to the number of carbon atoms of the monomer. Three PHA materials, Polyhydroxybutyrate (PHB), a copolyester of 3-hydroxybutanoic acid (3-HB) and 3-hydroxyvaleric acid (3-HV) (PHBV), a copolyester of 3-hydroxybutanoic acid and 3-hydroxyhexanoic acid (PHBHHx), are currently most used. The purified PHB is similar to thermoplastic plastics in certain properties, is brittle, has the elongation at break of only 5 percent, has high melting point, is difficult to process, and greatly limits the application of the PHB, and the incorporation of 3HV monomer in the PHB can reduce the hardness, the strength and the brittleness of the PHB without reducing the decomposition temperature, and has stronger elasticity than the PHB, thereby having great improvement effect on the properties of materials. Therefore, the PHBV is expected to replace the traditional petroleum-based plastic to become a new-generation green environment-friendly sustainable development material. It can be widely applied to the fields of degradable packaging materials, biomedical materials, controllable slow-release drug carriers and the like.
Disclosure of Invention
In view of the above, the present invention aims to provide a Rhodococcus pyridinivorans strain and its application in the production of PHBV, wherein the Rhodococcus pyridinivorans is named Rhodococcus pyridinivorans (Rhodococcus pyridinivorans) P23, which is synthesized into PHBV by using terephthalic acid as a sole carbon source, wherein the terephthalic acid is a raw material for producing ethylene terephthalate, is cheap and easy to obtain, the content of 3-HV monomer in the produced PHBV exceeds 60%, the weight average molecular weight is higher than 600kDa, and the physical and thermodynamic properties are excellent, so the present invention has good application prospects.
The invention adopts the specific technical scheme that:
the invention provides Rhodococcus pyridinivorans named Rhodococcus pyridinivorans P23 registered and preserved in China center for type culture Collection with the preservation number of CCTCC NO: m2019609.
Further, the 16S rDNA sequence of the strain is shown as SEQ ID NO: 1 is shown.
In a second aspect, the present invention provides the use of Rhodococcus pyridinovorans P23 in the production of PHBV, which is a copolymer of 3-hydroxybutyric acid and 3-hydroxyvaleric acid.
Further, in the above application, the synthetic substrate of PHBV is terephthalic acid and/or terephthalate. The chemical composition of the terephthalate is C8H4O4 & Rx, wherein R is Na, K, Li, Rb, Cs, Mg, Ca, Ba, Zn and other metals, and x is 1-2.
In a third aspect, the present invention provides a process for preparing PHBV, comprising the steps of: rhodococcus pyridinivorans (Rhodococcus pyridinivorans) P23 was cultured by fermentation, and PHBV was isolated and purified.
Preferably, in the method for preparing PHBV, the fermentation temperature is 25-40 ℃; the pH value of the fermentation system is 6.0-9.0; the dissolved oxygen of the fermentation system is 20-100 percent; the fermentation time is 24-96 hours.
Further, the method for the fermentation culture of Rhodococcus pyridinivorans (Rhodococcus pyridinivorans) P23 comprises the following steps:
(1) inoculating Rhodococcus pyridinivorans (Rhodococcus pyridinivorans) P23 into seed culture medium for fermentation culture;
(2) and (2) transferring the seeds in the step (1) to a fermentation culture medium for fermentation culture.
Preferably, in the method for fermentatively culturing Rhodococcus pyridinivorans (Rhodococcus pyridinivorans) P23, the seed culture medium in step (1) is 2216E medium containing 5.0g of peptone, 1.0g of yeast powder, 0.1g of ferric citrate, 19.45g of sodium chloride, 5.98g of magnesium chloride, 3.24g of sodium sulfate, 1.8g of calcium chloride, 0.55g of potassium chloride, 0.16g of sodium carbonate, 0.08g of potassium bromide, 0.034g of strontium chloride, 0.022g of boric acid, 0.004g of sodium silicate, 0.0024g of sodium fluoride, 0.0016g of sodium nitrate, 0.008g of disodium hydrogen phosphate, and is adjusted to 1 liter with water and has a pH value of 7.6 +/-0.2; and/or the presence of a gas in the gas,
the fermentation medium in the step (2) consists of 5.0-50.0g of sodium chloride, 1.0-10.0g of magnesium chloride hexahydrate, 0.1-2.0g of calcium chloride, 0.5-5.0g of sodium sulfate, 0.1-5.0g of ammonium chloride, 0.1-20.0g of monopotassium phosphate, 0.5-10.0g of potassium chloride, 0.5-3.0 ml of trace elements, 5.0-30.0g of terephthalic acid and/or terephthalate and water, and the volume is fixed to 1 liter by using water; and/or the presence of a gas in the gas,
and (3) inoculating the seeds cultured in the step (1) in the step (2) into a fermentation medium in an inoculation amount of 1%.
More preferably, the trace elements are prepared as follows: mixing 1.0-10.0g of ethylenediamine tetraacetic acid, 2.0-50.0mg of boric acid, 2.0-50.0mg of manganese chloride tetrahydrate, 1.0-5.0g of ferrous sulfate heptahydrate, 100.0-1000.0mg of cobalt chloride hexahydrate, 10.0-100.0mg of nickel chloride hexahydrate, 2.0-50.0mg of copper chloride dihydrate, 2.0-50.0mg of zinc sulfate heptahydrate, 2.0-50.0mg of sodium molybdate dihydrate and 0.5mol/L of hydrochloric acid aqueous solution, and supplementing to 1 liter with 0.5mol/L of hydrochloric acid aqueous solution.
Further, the PHBV is separated and purified by centrifuging after fermentation is finished, extracting the bacteria by chloroform after freeze drying, filtering the bacteria, performing rotary evaporation concentration, and precipitating by cold methanol to obtain the PHBV.
The invention has the beneficial effects that: experiments show that Rhodococcus pyridinovorans P23 provided by the invention can efficiently synthesize PHBV in a mineral salt culture medium by taking terephthalic acid and/or terephthalate as a unique carbon source, the content of 3-HV monomer in the synthesized PHBV exceeds 60%, the weight average molecular weight is higher than 600kDa, the physical property and the thermodynamic property are excellent, and the application prospect is good.
Drawings
FIG. 1 is a transmission electron micrograph of Rhodococcus pyridinovorans (Rhodococcus pyridinovorans) P23;
FIG. 2 shows the phylogenetic tree analysis of the rDNA sequence of Rhodococcus pyridinivorans (Rhodococcus pyridinivorans) P2316S;
FIG. 3 is a PHBV NMR spectrum synthesized by Rhodococcus pyridinovorans P23;
FIG. 4 is a PHBV NMR spectrum synthesized by Rhodococcus pyridinovorans P23;
FIG. 5 is the integral ratio of PHBV monomer 3-hydroxyvaleric acid and 3-hydroxybutyric acid methine hydrogen synthesized by Rhodococcus pyridinivorans P23.
Detailed Description
Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
The experimental procedures used in the examples described below are all conventional.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1
Separation and identification of strains:
diluting the obtained Pacific deep sea sediment with sterile water, coating 2216E solid agar plate, selecting single colony, streaking, purifying and culturing to obtain a strain capable of observing the white particle production in the strain under a transmission electron microscope, and naming the strain as P23. As shown in FIG. 1, the transmission electron micrograph of the cells of the strain P23 showed that the white particles in the cells were PHBV. Extracting the genome of the strain, and performing whole genome sequencing to obtain a 16S rDNA sequence shown as SEQ ID NO: 1 is shown. The 16S rDNA was aligned at https:// www.ezbiocloud.net/website and found to have a 99.93% similarity to the standard strain Rhodococcus pyridinivorans DSM 44555, indicating that strain P23 is Rhodococcus pyridinivorans (Rhodococcus pyridinivorans). A phylogenetic tree based on the 16S rDNA of the strain was also constructed (FIG. 2). The strain is preserved in China Center for Type Culture Collection (CCTCC) in 2019, 8 and 7 months, and the address is as follows: China-Wuhan university, zip code 430072, its deposit number is: CCTCC NO: m2019609.
2216E solid agar used for separating and purifying the bacterial strain contains 5.0g of peptone, 1.0g of yeast powder, 0.1g of ferric citrate, 19.45g of sodium chloride, 5.98g of magnesium chloride, 3.24g of sodium sulfate, 1.8g of calcium chloride, 0.55g of potassium chloride, 0.16g of sodium carbonate, 0.08g of potassium bromide, 0.034g of strontium chloride, 0.022g of boric acid, 0.004g of sodium silicate, 0.0024g of sodium fluoride, 0.0016g of sodium nitrate, 0.008g of disodium hydrogen phosphate and 20g of agar per liter of water, and the pH value is 7.6 +/-0.2.
Example 2
The PHBV is synthesized by utilizing Rhodococcus pyridinivorans (Rhodococcus pyridinivorans) P23 through fermentation by using terephthalic acid as a substrate:
rhodococcus pyridinivorans (Rhodococcus pyridinivorans) P23 was inoculated into 2216E liquid medium for test tube fermentation culture, and cultured seeds were inoculated at 1% to the fermentation medium. The fermentation medium comprises the following components: each liter contains 26.0g of sodium chloride, 5.0g of magnesium chloride hexahydrate, 1.06g of calcium chloride, 4.0g of sodium sulfate, 0.3g of ammonium chloride, 0.1g of monopotassium phosphate, 0.5g of potassium chloride, 1 ml of trace elements and 10g of terephthalic acid, and the volume is fixed to 1 liter by water. Adjusting the pH value to 7.0, the culture temperature to 30 ℃, the rotating speed of a shaking table to 180 r/min, and the fermentation time to 48 hours. The fermentation was tested in a 500mL Erlenmeyer flask, and the liquid loading was 150 mL. And after the fermentation is finished, centrifugally collecting thalli, extracting the thalli by using chloroform after freeze-drying the thalli, filtering the thalli, and precipitating the thalli by using cold methanol to obtain PHBV. The dry weight of the bacteria is 2.14g/L, the yield of PHBV is 0.32g/L, and the PHBV accounts for 15.0 percent of the dry weight of the bacteria. The PHBV is dissolved in deuterated chloroform, and hydrogen and carbon spectra are measured by 400M nuclear magnetic resonance spectrometer, and the carbon and hydrogen assignments of two monomers, namely 3-hydroxyvaleric acid and 3-hydroxybutyric acid, are shown in figures 3 and 4. Using the integration of the synthesized PHBV monomer, 3-hydroxyvaleric acid and 3-hydroxybutyric acid, the 3-hydroxyvaleric acid monomer content was found to be 75.2% (FIG. 5).
Example 3
Physical characterization of the PHBV synthesized by Rhodococcus pyridinivorans P23 fermentation:
taking a 10mg PHBV sample to carry out differential scanning calorimetry analysis, precooling the sample to-100 ℃, heating to 200 ℃ at the speed of 10 ℃/min and recording, quenching to-100 ℃, heating to 200 ℃ again at the speed of 10 ℃/min and recording. The PHBV melting temperature was found to be 101.0 ℃ and the glass transition temperature to-14.4 ℃. The tensile mechanics experiment of the dumbbell-shaped diaphragm made of PHBV with the thickness of 0.6mm and the length and width of 35 multiplied by 6mm shows that the tensile strength of the PHBV is 1.0Mpa and the elongation at break is 15%. 10mg of PHBV sample was dissolved in chloroform and analyzed by gel permeation chromatography for molecular weight determination, which showed a weight average molecular weight of 820 kDa.
Example 4
The PHBV is synthesized by fermenting Rhodococcus pyridinivorans (Rhodococcus pyridinivorans) P23 with disodium terephthalate as a substrate:
rhodococcus pyridinivorans (Rhodococcus pyridinivorans) P23 was inoculated into 2216E liquid medium for fermentation culture, and cultured seeds were inoculated into the fermentation medium at 1%. The fermentation medium comprises the following components: each liter of the solution contains 15.0g of sodium chloride, 2.0g of magnesium chloride hexahydrate, 0.5g of calcium chloride, 3.0g of sodium sulfate, 0.5g of ammonium chloride, 1.0g of monopotassium phosphate, 0.5g of potassium chloride, 1 ml of trace elements and 20g of disodium terephthalate, and the volume is fixed to 1 liter by water. Adjusting the pH value to 7.0, the culture temperature to 30 ℃, the rotating speed of a shaking table to 180 r/min, and the fermentation time to 72 hours. The fermentation was tested in a 500mL Erlenmeyer flask, and the liquid loading was 150 mL. And after the fermentation is finished, centrifugally collecting thalli, extracting the thalli by using chloroform after freeze-drying the thalli, filtering the thalli, and precipitating the thalli by using cold methanol to obtain PHBV. The dry weight of the bacteria is 2.65g/L, the yield of PHBV is 0.63g/L, and the PHBV accounts for 23.8 percent of the dry weight of the bacteria.
Although the embodiments have been described, once the basic inventive concept is obtained, other variations and modifications of these embodiments can be made by those skilled in the art, so that the above embodiments are only examples of the present invention, and not intended to limit the scope of the present invention, and all equivalent structures or equivalent processes using the contents of the present specification and drawings, or any other related technical fields, which are directly or indirectly applied thereto, are included in the scope of the present invention.
Sequence listing
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China ocean mineral resources research and development association (China ocean affairs administration)
<120> Rhodococcus pyridinivorans and application thereof in production of PHBV
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Claims (10)
1. The Rhodococcus pyridinivorans is named Rhodococcus pyridinivorans (Rhodococcus pyridinivorans) P23 and is registered and preserved in China center for type culture Collection with the preservation number of CCTCC NO: m2019609.
2. The Rhodococcus pyridinivorans strain of claim 1 having a 16S rDNA sequence as set forth in SEQ ID NO: 1 is shown.
3. Use of Rhodococcus pyridinivorans P23 for the production of PHBV, which is a copolymer of 3-hydroxybutyric acid and 3-hydroxyvaleric acid.
4. Use according to claim 3, characterized in that the synthetic substrate of PHBV is terephthalic acid and/or terephthalate.
5. A process for the preparation of PHBV, characterized in that it comprises the following steps: rhodococcus pyridinivorans (Rhodococcus pyridinivorans) P23 was cultured by fermentation, and PHBV was isolated and purified.
6. The method for preparing PHBV according to claim 5, wherein the fermentation temperature is 25-40 ℃; the pH value of the fermentation system is 6.0-9.0; the dissolved oxygen of the fermentation system is 20-100%, and the fermentation time is 24-96 hours.
7. The process for preparing PHBV according to claim 5 or 6, wherein said fermentation culture of Rhodococcus pyridinivorans P23 is performed by:
(1) inoculating Rhodococcus pyridinivorans (Rhodococcus pyridinivorans) P23 into seed culture medium for fermentation culture;
(2) and (2) transferring the seeds in the step (1) to a fermentation culture medium for fermentation culture.
8. The method for preparing PHBV according to claim 7, wherein the seed medium in step (1) is 2216E medium containing 5.0g of peptone, 1.0g of yeast powder, 0.1g of ferric citrate, 19.45g of sodium chloride, 5.98g of magnesium chloride, 3.24g of sodium sulfate, 1.8g of calcium chloride, 0.55g of potassium chloride, 0.16g of sodium carbonate, 0.08g of potassium bromide, 0.034g of strontium chloride, 0.022g of boric acid, 0.004g of sodium silicate, 0.0024g of sodium fluoride, 0.0016g of sodium nitrate, 0.008g of disodium hydrogen phosphate, and the volume is adjusted to 1L with water, and the pH value is 7.6 +/-0.2; and/or the presence of a gas in the gas,
the fermentation medium in the step (2) consists of 5.0-50.0g of sodium chloride, 1.0-10.0g of magnesium chloride hexahydrate, 0.1-2.0g of calcium chloride, 0.5-5.0g of sodium sulfate, 0.1-5.0g of ammonium chloride, 0.1-20.0g of monopotassium phosphate, 0.5-10.0g of potassium chloride, 0.5-3.0 ml of trace elements, 5.0-30.0g of terephthalic acid and/or terephthalate and water, and the volume is fixed to 1 liter by using water; and/or the presence of a gas in the gas,
and (3) inoculating the seeds cultured in the step (1) in the step (2) into a fermentation medium in an inoculation amount of 1%.
9. The process for the preparation of PHBV according to claim 8 wherein said trace elements are prepared as follows: mixing 1.0-10.0g of ethylenediamine tetraacetic acid, 2.0-50.0mg of boric acid, 2.0-50.0mg of manganese chloride tetrahydrate, 1.0-5.0g of ferrous sulfate heptahydrate, 100.0-1000.0mg of cobalt chloride hexahydrate, 10.0-100.0mg of nickel chloride hexahydrate, 2.0-50.0mg of copper chloride dihydrate, 2.0-50.0mg of zinc sulfate heptahydrate, 2.0-50.0mg of sodium molybdate dihydrate and 0.5mol/L of hydrochloric acid aqueous solution, and supplementing to 1 liter with 0.5mol/L of hydrochloric acid aqueous solution.
10. The method for preparing PHBV according to claim 5 or 6, wherein the PHBV is obtained by centrifugation after fermentation, freeze-drying the cells, extracting with chloroform, filtering the cells, rotary evaporation and concentration, and precipitation with cold methanol.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115322926A (en) * | 2022-07-28 | 2022-11-11 | 广西民族大学 | Rhodococcus pyridinivorans and application thereof in repairing continuous cropping and continuous cropping soil |
| CN115386520A (en) * | 2022-08-26 | 2022-11-25 | 广东海洋大学 | Rhodococcus pyridinivorans RL-GZ01 strain and application thereof |
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| CN115322926A (en) * | 2022-07-28 | 2022-11-11 | 广西民族大学 | Rhodococcus pyridinivorans and application thereof in repairing continuous cropping and continuous cropping soil |
| CN115322926B (en) * | 2022-07-28 | 2024-02-09 | 广西民族大学 | Rhodococcus pyridine and application thereof in repairing continuous cropping soil |
| CN115386520A (en) * | 2022-08-26 | 2022-11-25 | 广东海洋大学 | Rhodococcus pyridinivorans RL-GZ01 strain and application thereof |
| CN115386520B (en) * | 2022-08-26 | 2023-11-14 | 广东海洋大学 | Rhodococcus pyridine-philic RL-GZ01 strain and application thereof |
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