CN113265357A - Vibrio costalis for producing polyhydroxyalkanoate and application thereof - Google Patents
Vibrio costalis for producing polyhydroxyalkanoate and application thereof Download PDFInfo
- Publication number
- CN113265357A CN113265357A CN202110571791.3A CN202110571791A CN113265357A CN 113265357 A CN113265357 A CN 113265357A CN 202110571791 A CN202110571791 A CN 202110571791A CN 113265357 A CN113265357 A CN 113265357A
- Authority
- CN
- China
- Prior art keywords
- vibrio
- polyhydroxyalkanoate
- carbon source
- culture medium
- hydroxybutyrate
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Classifications
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/62—Carboxylic acid esters
- C12P7/625—Polyesters of hydroxy carboxylic acids
Abstract
The invention discloses a vibrio costigena for producing polyhydroxyalkanoate and application thereof. The Vibrio costicola has a strain number of Salinivibrio costicola-TGB-10, and the preservation number of the Vibrio costicola is CGMCC No.21104 in the China Committee for culture Collection of microorganisms. The Vibrio costigena of the present invention is used in any one of the following applications: 1) producing polyhydroxyalkanoate; 2) preparing a product for producing polyhydroxyalkanoate. The strain can utilize a saccharide raw material or volatile fatty acid as a carbon source to quickly grow and accumulate the poly-3-hydroxybutyrate; the raw materials of the saccharides and the propionic acid can be used as a mixed carbon source to rapidly grow and accumulate the poly (3-hydroxybutyrate-co-3-hydroxyvalerate); the invention can reduce the fermentation cost of PHBV, obtain the polyhydroxy fatty acid ester with various 3-hydroxyvaleric acid monomer contents, and has great significance for promoting the industrial production of PHBV.
Description
Technical Field
The invention belongs to the fields of microbiology and fermentation engineering, and relates to vibrio costigena for producing polyhydroxyalkanoate and application thereof.
Background
Traditional petroleum-derived plastic materials are important commodities for improving comfort and quality of life. These plastics are not degradable, leading to a constant accumulation in the environment, and have become an increasingly serious environmental pollution problem. In order to solve the increasingly serious problem of white garbage pollution, nine departments, such as 2020, 7 months, national development and transformation committee, ecological environment department and the like, jointly issue a notice about the work of strengthening and propelling plastic pollution control, and propose that from 2021, 1 month and 1 day, the use of non-degradable plastic shopping bags in markets, supermarkets, drug stores, bookstores and other places in built-up areas of direct prefecture cities, provincial cities and planned single-row cities, the production and sale of disposable plastic swabs and disposable foamed plastic tableware are prohibited nationwide, and the use of non-degradable disposable plastic straws is prohibited in the catering industry.
Polyhydroxyalkanoate (PHA) is a large class of biodegradable materials with good application prospects, and can be obtained by fermenting renewable biomass resources with microorganisms. PHA has material properties similar to those of petroleum-derived plastic materials, while having excellent properties such as biorenewability, biodegradability and biocompatibility, etc., which are lacking in many petroleum-derived plastic materials. Poly-3-hydroxybutyrate (PHB) is the simplest and earliest discovered PHA material. PHB has a wide range of applications due to its excellent biodegradability and biocompatibility, but it also has some inherent disadvantages, such as brittleness, difficulty in processing, etc., which limit the range of applications of PHB. The introduction of 3-hydroxyvalerate (3-hydroxyvalerate, abbreviated as 3HV) unit into the polymer to generate poly (3-hydroxybutyrate-co-3-hydroxyvalerate) is an effective way to improve the material performance. Compared with the PHB homopolymer, the PHBV copolyester has better toughness, the processing window is correspondingly widened, good biodegradability and biocompatibility are kept, and the material performance can be adjusted in a large range along with the different content of the 3HV monomer in the copolyester.
Disclosure of Invention
The invention aims to provide vibrio costigena for producing polyhydroxyalkanoate and application thereof.
The vibrio costicola is Salinivibrio costicola, the strain number of the vibrio costicola is Salinivibrio costicola-TGB-10, and the preservation number of the vibrio costicola in the common microorganism center of China Committee for culture Collection of microorganisms is CGMCC No. 21104.
The vibrio costicola is called S.costicola TGB10 for short.
The Vibrio costigena of the present invention is used in any one of the following applications:
1) producing polyhydroxyalkanoate;
2) preparing a product for producing polyhydroxyalkanoate.
In the application of the vibrio costigena, the carbon source used for producing the polyhydroxyalkanoate is a carbohydrate raw material.
In the application of the vibrio costigena, the carbon source used for producing the polyhydroxyalkanoate is volatile fatty acid.
In the application of the vibrio costigena, the carbon source for producing the polyhydroxyalkanoate is a carbohydrate raw material and volatile fatty acid.
In the above application of Vibrio costata, the saccharide raw material is at least one selected from glucose, maltose and soluble starch.
In the above application of Vibrio costata, the volatile fatty acid is at least one selected from the group consisting of acetic acid, propionic acid and butyric acid.
In the above application of Vibrio costata, the polyhydroxyalkanoate comprises poly-3-hydroxybutyrate (PHB) and/or poly (3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV). The poly (3-hydroxybutyrate-co-3-hydroxyvalerate) is a copolyester containing two monomer components, namely 3-hydroxybutyrate and 3-hydroxyvalerate.
The present invention further provides a method for producing polyhydroxyalkanoate, comprising the steps of: fermenting and culturing the vibrio costigena by using a culture medium containing the carbon source to obtain a fermentation product; obtaining the polyhydroxyalkanoate from the fermentation product.
In the above method, the culture medium comprises an artificial seawater culture medium or a natural seawater culture medium;
the artificial seawater culture medium consists of a solute and a solvent, wherein the solvent is artificial seawater, the concentration of the solute is 1-5 g/L of peptone, 0-5 g/L of yeast powder and 10-30 g/L of a carbon source;
the natural seawater culture medium is composed of a solute and a solvent, the solvent is natural seawater, the solute and the concentration thereof are 5-20 g/L of corn steep liquor, and the carbon source is 10-30 g/L.
In the invention, the artificial seawater comprises the following components in concentration: 27.5g/L of sodium chloride, 0.7g/L of potassium chloride, 2.5g/L of magnesium chloride, 3.3g/L of magnesium sulfate, 1g/L of calcium chloride and 0.2g/L of sodium bicarbonate, wherein the solvent of the artificial seawater is water;
the natural seawater is obtained from the offshore of new coastal area in Tianjin City.
In the above method, the artificial seawater culture medium or the natural seawater culture medium further comprises 15g/L agar powder component to prepare a solid culture medium.
In the above method, when the carbon source is a saccharide raw material or the volatile fatty acid, the obtained polyhydroxyalkanoate is poly-3-hydroxybutyrate.
In the above method, when the carbon source is a mixture of the saccharide raw material and the propionic acid, the obtained polyhydroxyalkanoate is poly (3-hydroxybutyrate-co-3-hydroxyvalerate).
The invention further provides a culture medium, which is the artificial seawater culture medium or the natural seawater culture medium.
The invention has the following advantages:
1. the strain can utilize sugar raw materials (glucose, maltose, soluble starch and the like) as carbon sources to rapidly grow and accumulate the poly-3-hydroxybutyrate, the proportion of the polyester to the dry weight of bacteria can reach 80 percent at most, and the yield in a shake flask can reach 7 g/L.
2. The strain can utilize volatile fatty acid (acetic acid, propionic acid, butyric acid and the like) as a carbon source to rapidly grow and accumulate poly-3-hydroxybutyrate, the proportion of the polyester to the dry weight of bacteria can reach 30 percent at most, and the yield in a shake flask can reach 0.81 g/L.
3. The strain can utilize sugar raw materials (glucose, maltose, soluble starch and the like) and propionic acid (the concentration is 2g/L-8g/L) as a mixed carbon source to rapidly grow and accumulate poly (3-hydroxybutyrate-co-3-hydroxyvalerate), the proportion of polyester to the dry weight of bacteria can reach 60 percent at most, the yield in a shake flask can reach 4g/L, and the highest 3HV monomer content can reach 93 percent.
4. The invention can reduce the fermentation cost of PHBV, obtain the polyhydroxy fatty acid ester with various 3-hydroxyvaleric acid monomer contents, and has great significance for promoting the industrial production of PHBV.
Deposit description
Classification nomenclature of biological materials: vibrio costata.
Latin name of biomaterial: salinivibrio costicola.
According to the biological materials (strains): salinivibrio costicola-TGB-10.
The preservation unit is called as follows: china general microbiological culture Collection center.
The preservation unit is abbreviated as: CGMCC.
Address: xilu No. 1 Hospital No. 3, Beijing, Chaoyang, North.
The preservation date is as follows: 2020.11.05.
the preservation number is: CGMCC No. 21104.
Drawings
FIG. 1 is a TEM photograph of a Vibrio costatum strain in TYS medium supplemented with glucose as a carbon source in example 1 of the present invention.
FIG. 2 is a transmission electron micrograph of a Vibrio costatum strain in TYS medium according to example 1 of the present invention.
Detailed Description
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
In the quantitative experiments in the following examples, three replicates were set up and the results averaged. Polyhydroxyalkanoate standards are available from Sigma-Aldrich under the product designation 403121, and are known as poly (3-hydroxybutyrate-co-3-hydroxyvalerate) with a 3-hydroxybutyrate monomer content of 88 mol% and a 3-hydroxyvalerate monomer content of 12 mol%.
The method of freeze-drying in the following examples is as follows:
after carrying out fermentation culture on microorganisms, taking fermentation liquor, centrifuging at 10000rpm for 10min, discarding supernatant, then using deionized water to resuspend thalli for washing, centrifuging at 10000rpm for 10min again to collect thalli, placing a centrifugal tube containing washed thalli precipitates in a freezing vacuum drier for freezing for 2h at-20 ℃, and then placing the centrifugal tube in the freezing vacuum drier for freeze-drying for 10h to obtain a freeze-dried product.
The dry cell weight is measured in the following examples as dry cell weight per liter of fermentation broth. The unit of cell dry weight is g/L. Dry cell weight (CDW) (weight of the centrifugal tube after freeze drying-weight of the original hollow centrifugal tube)/amount of fermentation liquor; the weight of the centrifugal tube after freeze drying and the weight of the original hollow centrifugal tube are both g; the fermentation liquor taking unit is L.
The method for detecting the content of the bacterial polyhydroxyalkanoate in the following examples: performing esterification reaction on the freeze-dried product, and then calculating by measuring the content of the product after the esterification reaction;
esterification reaction: putting 30-40mg of freeze-dried product into an esterification tube, adding 2mL of chloroform and 2mL of esterification solution (the esterification solution is obtained by adding 15mL of concentrated sulfuric acid and 0.5g of benzoic acid into 500mL of methanol), mixing uniformly, covering and sealing, and esterifying at high temperature of 100 ℃ for 4 hours; cooling to room temperature, adding 1mL of deionized water, fully oscillating and uniformly mixing by using a vortex oscillator, standing and layering; after the chloroform phase was completely separated from the water, 1. mu.L of the chloroform phase was taken for gas chromatography.
About 20mg of poly (3-hydroxybutyrate-co-3-hydroxyvalerate) was taken and subjected to esterification reaction in the same manner to obtain a standard.
Gas chromatography analysis parameters: using HP 6890 gas chromatograph, the chromatographic column is HP-5 capillary column, the column length is 30m, the inner diameter is 320 μm, and the stationary phase is phenyl methyl polysiloxane with thickness of 25 nm; the detector is a Flame Ionization Detector (FID); high-purity nitrogen is used as carrier gas, hydrogen is used as fuel gas, and air is used as combustion-supporting gas;
the conditions for gas chromatography were as follows:
(1) column temperature: starting at 80 ℃, and staying for 1.5 min; heating to 140 deg.C at a rate of 30 deg.C/min, and standing for 0 min; heating to 220 deg.C at 40 deg.C/min, and standing for 1 min. The total time was 6.5 min.
(2) Column pressure: starting at 10psi, and staying for 1.5 min; the pressure was increased to 20psi at a rate of 2.5psi/min, and the residence time was 0.5 min. (psi is pressure units, i.e., pounds per square inch, 1psi 6.89476kPa)
(3) A sample inlet: the temperature was 200 ℃ and the split mode was used with a split ratio of 30.
(4) A detector: the temperature was 220 ℃, the hydrogen flow rate was 30mL/min, and the air flow rate was 400 mL/min.
A microsyringe from Agilent was used in an amount of 1. mu.L, and the polymer was quantitatively analyzed by an internal standard method and quantified based on the peak area.
For gas chromatography, lyophilized cell samples were compared to poly (3-hydroxybutyrate-co-3-hydroxyvalerate) standards. The esterification reaction and the gas chromatography detection are carried out by adopting the steps, and the freeze-dried cell sample contains a signal with the peak position same as the position of the 3-hydroxybutyrate in the standard product, namely the polyhydroxy fatty acid ester accumulated in the thallus is shown to be poly-3-hydroxybutyrate. The freeze-dried cell sample contains signals with peak positions the same as those of 3-hydroxybutyric acid and 3-hydroxyvaleric acid in a standard sample, namely, the polyhydroxy fatty acid ester accumulated in the thalli is poly (3-hydroxybutyrate-co-3-hydroxyvalerate).
The poly-3-hydroxybutyrate production algorithm was: PHB yield (PHB peak area in sample/internal standard peak area in sample) × [ (internal standard peak area in standard/PHB peak area in standard) × (standard mass × 0.866) ]/sample esterification mass × cell dry weight
The poly (3-hydroxybutyrate-co-3-hydroxyvalerate) production algorithm is: PHBV yield + PHV yield, wherein:
PHB yield (PHB peak area in sample/internal standard peak area in sample) × [ (internal standard peak area in standard/PHB peak area in standard) × (standard mass × 0.866) ]/sample esterification mass × cell dry weight
PHV yield ═ PHV peak area in sample/internal standard peak area in sample × [ (internal standard peak area in standard/PHV peak area in standard) × (standard mass × 0.134) ]/sample esterification mass × cell dry weight
The polymer content is defined as the ratio of polymer to dry cell weight, polymer content ═ polymer yield/dry cell weight × 100%.
The composition of the TYS liquid medium was as follows: the nutrient solution consists of solute and solvent, wherein the pH value of the solution is 7.5, the solvent is artificial seawater, the concentration of the solute is 5g/L of peptone, and the concentration of yeast powder is 1 g/L. The artificial seawater is composed of solute and solvent, the solvent is water, and the solute and the concentration thereof are as follows: 27.5g/L of sodium chloride, 0.7g/L of potassium chloride, 2.5g/L of magnesium chloride, 3.3g/L of magnesium sulfate, 1g/L of calcium chloride and 0.2g/L of sodium bicarbonate.
TYS liquid medium containing 20g/L glucose: the solution consists of solute and solvent, the pH is 7.5, the solvent is the artificial seawater, the solute and the concentration thereof are 5g/L of peptone, 1g/L of yeast powder and 20g/L of glucose.
Liquid TYS medium containing 20g/L maltose: the solution consists of solute and solvent, the pH is 7.5, the solvent is the artificial seawater, the solute and the concentration thereof are 5g/L of peptone, 1g/L of yeast powder and 20g/L of maltose.
Liquid TYS medium containing 20g/L soluble starch: the solution consists of solute and solvent, the pH is 7.5, the solvent is the artificial seawater, the solute and the concentration thereof are 5g/L of peptone, 1g/L of yeast powder and 20g/L of soluble starch.
Liquid TYS medium containing 20g/L acetic acid: the solution consists of solute and solvent, the pH is 7.5, the solvent is the artificial seawater, the solute and the concentration thereof are 5g/L of peptone, 1g/L of yeast powder and 20g/L of acetic acid.
Liquid TYS medium containing 10g/L propionic acid: the solution consists of solute and solvent, the pH is 7.5, the solvent is the artificial seawater, the solute and the concentration thereof are 5g/L of peptone, 1g/L of yeast powder and 10g/L of propionic acid.
Liquid TYS medium containing 10g/L butyric acid: the solution consists of solute and solvent, the pH is 7.5, the solvent is the artificial seawater, and the solute and the concentration thereof are 5g/L of peptone, 1g/L of yeast powder and 10g/L of butyric acid.
Liquid TYS medium containing 20g/L glucose and different concentrations of propionic acid (2g/L, 4g/L, 6g/L or 8 g/L): the solution consists of solute and solvent, the pH is 7.5, the solvent is the artificial seawater, and the concentration of the solute is 5g/L of peptone, 1g/L of yeast powder, 20g/L of glucose, 2g/L, 4g/L, 6g/L or 8g/L of propionic acid.
Liquid TYS medium containing 20g/L maltose and different concentrations of propionic acid (2g/L, 4g/L, 6g/L, 8 g/L): the solution consists of solute and solvent, the pH is 7.5, the solvent is the artificial seawater, and the concentration of the solute is 5g/L of peptone, 1g/L of yeast powder, 20g/L of maltose, 2g/L, 4g/L, 6g/L or 8g/L of propionic acid.
Liquid TYS medium containing 20g/L of soluble starch and different concentrations of propionic acid (2g/L, 4g/L, 6g/L or 8 g/L): the solution consists of solute and solvent, the pH is 7.5, the solvent is the artificial seawater, the solute and the concentration thereof are 5g/L of peptone, 1g/L of yeast powder, 20g/L of soluble starch, 2g/L, 4g/L, 6g/L or 8g/L of propionic acid.
The composition of the TYS solid medium was as follows: the artificial seawater desalination device comprises a solute and a solvent, wherein the solvent is the artificial seawater, and the solute and the concentration thereof are 5g/L of peptone, 1g/L of yeast powder and 15g/L of agar.
The composition of the natural seawater culture medium containing 20g/L glucose is as follows: the solute and the solvent are composed of the solute and the solvent, the solvent is natural seawater, is particularly taken from the offshore of a new coastal region in Tianjin, and the concentration of the solute are 15g/L of corn steep liquor and 20g/L of glucose.
Example 1: isolation and characterization of Salinivibrio costicola-TGB-10
1. Isolation of Salinivibrio costicola-TGB-10:
and collecting a water sample of a salt pond of a new coastal area in Tianjin. Adding 1mL of water sample into a TYS liquid culture medium containing 20g/L of acetic acid, culturing at 30 ℃ and 200rpm for 24h, streaking the TYS solid culture medium containing 20g/L of acetic acid by using a sterilized inoculating loop, culturing upside down for 24h, picking a monoclonal, purifying on a TYS solid culture medium plate, determining the monoclonal to be pure bacteria, transferring to a slope for short-term storage at 4 ℃, transferring to a 25% glycerol tube, and storing at-80 ℃ for long-term storage. One of the separated and purified strains TGB-10 is Salinivibrio costicola-TGB-10.
2. Identification of Salinivibrio costicola-TGB-10:
(1) observation of culture characteristics of Strain Salinivibrio costicola-TGB-10
The strain Salinivibrio costicola-TGB-10 is inoculated on a TYS culture medium and cultured for 10 hours at 30 ℃ to form a round colony which is 1.2-1.8mm, smooth in edge, convex in surface and milky in color. The cells of the strain Salinivibrio costicola-TGB-10 were rod-shaped, oblong and slightly curved as observed by transmission electron microscopy.
(2) Analysis of physiological and biochemical characteristics
The strain Salinivibrio costicola-TGB-10 is gram-negative bacteria and can grow on TYS and LB culture media, and the most suitable culture medium is TYS culture medium; the growth temperature is 20-45 ℃, and the optimal temperature is 30 ℃; the pH range of growth is 6-10, and the optimum pH is 7; the range of sodium chloride growth is 1-20% (w/v), and the optimum sodium chloride concentration is 3% (w/v).
(3)16s rDNA sequence analysis
The 16S rDNA sequence of the strain Salinivibrio costicola-TGB-10 is as follows:
AGAGTTTGATCCTGGCTCAGATTGAACGCTGGCGGCAGGCCTAACACATGCAAGTCGAGCGGAAACGGCAGTATTGAAGCTTCGGTGGATTTACTGGACGTCGAGCGGCGGACGGGTGAGTAACGGCTGGGAACCTGCCCTGACGAGGGGGATAACCGTTGGAAACGACGGCTAATACCGCATAATGTCTACGGACCAAAGGTGGCCTCTACATGTAAGCTATCGCGTCGGGATGGGCCCAGTTAGGATTAGCTAGTTGGTAAGGTAACGGCTTACCAAGGCGACGATCCTTAGCTGGTTTGAGAGGATGATCAGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGGGAGACCCTGATGCAGCCATGCCGCGTGTGTGAAGAAGGCCTTCGGGTTGTAAAGCACTTTCAGCAGTGAGGAAGGTAGTGTACTTAATACGTGCATTGCTTGACGTTAGCTGCAGAAGAAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCGAGCGTTAATCGGAATTACTGGGCGTAAAGCGCATGCAGGCGGTTTGTTAAGTCAGATGTGAAAGCCCGGGGCTCAACCTCGGAACCGCATTTGAAACTGGCAGGCTAGAGTCTTGTAGAGGGGGGTAGAATTTCAGGTGTAGCGGTGAAATGCGTAGAGATCTGAAGGAATACCAGTGGCGAAGGCGGCCCCCTGGACAAAGACTGACGCTCAGATGCGAAAGCGTGGGTAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGTCTACTTGGAGGCTGAGGTTTAAGACTTTGGCTTTCGGAGCTAACGCATTAAGTAGACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTACTCTTGACATCCAGCGAATCCTTTAGAGATAGAGGCGTGCCTTCGGGAGCGCTGAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTTGTGAAATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATCCTTGTTTGCCAGCACGTAATGGTGGGAACTCCAGGGAGACTGCCGGTGATAAACCGGAGGAAGGTGGGGACGACGTCAAGTCATCATGGCCCTTACGAGTAGGGCTACACACGTGCTACAATGGCAGATACAGAGGGCAGCGAGACAGCGATGTTAAGCGAATCCCTTAAAGTTTGTCGTAGTCCGGATTGGAGTCTGCAACTCGACTCCATGAAGTCGGAATCGCTAGTAATCGCAGATCAGAATGCTGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTGGGCTGCACCAGAAGTAGATAGCTTAACCTTCGGGAGGGCGTTTACCACGGTGTGGTTCATGACTGGGGTGAAGTCGTAACAAGGTAACC
the 16S rDNA sequence of the strain Salinivibrio costicola-TGB-10 is 1519bp, and gene sequence comparison analysis shows that the Salinivibrio costicola-TGB-10 has higher similarity with the gene sequence of the strain in the Salinivibrio genus, and the strain with the highest similarity is S.costicola M318 (99.94%).
The strain Salinivibrio costicola-TGB-10 of the invention was finally determined to be Vibrio costicola (Salinivibrio costicola) by combining the above morphological, physiological and biochemical characteristics, and the results of the genomic sequence determination.
The strain Salinivibrio costicola-TGB-10 has been deposited in the China general microbiological culture Collection center (CGMCC). Address: western road No. 1, north chen west road, north kyo, chaoyang, institute of microbiology, china academy of sciences, zip code 100101. The preservation date is 2020, 11, 05. The preservation number is CGMCC No. 21104. The strain name: vibrio costorum; latin name: salinivibrio costicola; according to the biological materials (strains): salinivibrio costicola-TGB-10.
Example 2: production of polyhydroxyalkanoates using glucose as carbon source
1. Preparation of seed liquid of Vibrio costicola TGB10 by aseptic operation
(1) Strain activation
Taking strain glycerine tube stored in refrigerator at-80 deg.C, streaking and inoculating to TYS solid culture medium plate, and culturing at 30 deg.C for 16 h.
(2) Preparation of seed liquid
And (3) picking single colonies from the plate which is subjected to the step (1), inoculating the single colonies into a TYS liquid culture medium, and performing shaking culture at 30 ℃ and 200rpm for 12 hours to obtain a seed solution.
2. The experiment was set up for two treatments: experimental and control groups.
Experimental groups: the seed solution obtained in step 1(2) was inoculated into a TYS liquid medium containing 20g/L glucose in an inoculum size of 2.5% (i.e., 1mL of seed solution, 39mL of medium), and cultured at 30 ℃ and 200rpm for 36 hours in a 250mL shake flask in a total liquid volume of 40mL to prepare a fermentation liquid.
Control group: the seed solution obtained in step 1(2) was inoculated into a TYS liquid medium in an inoculum size of 2.5% (1 mL of seed solution, 39mL of medium), and cultured at 30 ℃ and 200rpm for 36 hours in a 250mL shake flask to prepare a fermentation broth as a control.
3. And (3) putting all fermentation liquor into a centrifuge tube with the volume of 50mL, centrifuging at 10000rpm for 10min, discarding supernatant, then using deionized water to resuspend the thalli for washing, centrifuging at 10000rpm for 10min again to collect the thalli, putting the centrifuge tube filled with washed thalli precipitates into a freezing vacuum drier for freezing for 2h at-20 ℃, and then putting the centrifuge tube into a freezing vacuum drier for freeze-drying for 10h to obtain a freeze-dried product. Accurately weighing the weight of the centrifuge tube before adding the fermentation liquor and after freeze-drying, and calculating the dry weight of the cells.
4. Transferring the freeze-dried thallus into an esterification tube, weighing the dry weight (about 30-40mg) of the transferred thallus, adding 2mL of esterification solution and 2mL of chloroform, covering and sealing, and reacting in an oven at 100 ℃ for 4 h; cooling to room temperature, adding 1mL of deionized water, fully oscillating, standing and layering; after the chloroform phase and the water phase are completely separated, taking the chloroform phase and carrying out gas chromatography analysis;
a microsyringe from Agilent was used in an amount of 1. mu.L, and the polymer was quantitatively analyzed by an internal standard method and quantified based on the peak area.
The poly-3-hydroxybutyrate production algorithm was: PHB yield (PHB peak area in sample/internal standard peak area in sample) × [ (internal standard peak area in standard/PHB peak area in standard) × (standard mass × 0.866) ]/sample esterification mass × cell dry weight
By calculation, in an experimental group, the dry weight of the cell when glucose is used as a carbon source is 8.82g/L, the polyhydroxy fatty acid ester accumulated in the cell is poly-3-hydroxybutyrate, the yield is 6.84g/L, and the mass fraction of the polyhydroxy fatty acid ester in the dry weight of the cell is 77.52%.
In the control group, when glucose was not added, the dry weight of the cells was 1.43g/L, and no polyhydroxyalkanoate was produced.
Further, observation of the bacteria of example 2 after culture was carried out by transmission electron microscopy, and the results are shown in FIGS. 1 and 2, respectively, in which each scale is 200 nm. In FIG. 1, when glucose was used as a carbon source, a large amount of white polyhydroxyalkanoate particles were accumulated in the cells. In FIG. 2, when glucose was not added, a large amount of white polyhydroxyalkanoate particles were not observed in the cells.
Example 3: production of polyhydroxyalkanoates using maltose as carbon source
Specific operation according to example 2 of the present invention, except that the culture medium of the experimental group was a liquid TYS medium containing 20g/L maltose.
By calculation, in the experimental group, the cell dry weight of maltose as a carbon source is 7.28g/L, the polyhydroxyalkanoate ester accumulated in the cell is poly-3-hydroxybutyrate, the yield is 2.81g/L, and the mass fraction of the polyhydroxyalkanoate ester in the cell dry weight is 38.79%.
Example 4: production of polyhydroxyalkanoate by using soluble starch as carbon source
Specific operation according to example 2 of the present invention, except that the medium in the experimental group was a liquid TYS medium containing 20g/L of soluble starch.
By calculation, the dry weight of the cells when the soluble starch is used as a carbon source is 8.25g/L, the polyhydroxyalkanoate accumulated in the cells is poly-3-hydroxybutyrate, the yield is 3.28g/L, and the mass fraction of the polyhydroxyalkanoate accounts for 39.80% of the dry weight of the cells.
Example 5: production of polyhydroxyalkanoates using acetic acid as carbon source
Specific operation according to example 2 of the present invention, except that the medium in the experimental group was a liquid TYS medium containing 20g/L of acetic acid.
By calculation, the dry weight of the cells when acetic acid is used as a carbon source is 3.26g/L, the polyhydroxyalkanoate accumulated in the cells is poly-3-hydroxybutyrate, the yield is 0.21g/L, and the mass fraction of the polyhydroxyalkanoate accounting for the dry weight of the cells is 6.36%.
Example 6: production of polyhydroxyalkanoates using propionic acid as carbon source
Specific operation according to example 2 of the present invention, except that the medium in the experimental group was a liquid TYS medium containing 10g/L propionic acid. And 4, detecting the accumulation of the poly (3-hydroxybutyrate-co-3-hydroxyvalerate).
The PHBV yield algorithm is as follows: PHBV yield + PHV yield, wherein:
PHB yield (PHB peak area in sample/internal standard peak area in sample) × [ (internal standard peak area in standard/PHB peak area in standard) × (standard mass × 0.866) ]/sample esterification mass × cell dry weight
PHV yield ═ PHV peak area in sample/internal standard peak area in sample × [ (internal standard peak area in standard/PHV peak area in standard) × (standard mass × 0.134) ]/sample esterification mass × cell dry weight
By calculation, the cell dry weight of propionic acid as a carbon source is 2.20g/L, the polyhydroxyalkanoate accumulated in the cell is poly (3-hydroxybutyrate-co-3-hydroxyvalerate), the yield is 0.22g/L, the mass fraction of the polyhydroxyalkanoate in the cell dry weight is 9.80%, and the content of the 3-hydroxyvalerate monomer in the copolyester is 46.62 mol%.
Example 7: production of polyhydroxyalkanoates using butyric acid as carbon source
Specific operation according to example 2 of the present invention, except that the medium in the experimental group was a liquid TYS medium containing 10g/L of butyric acid.
By calculation, the dry weight of the cells when butyric acid is used as a carbon source is 2.49g/L, the polyhydroxyalkanoate accumulated in the cells is poly-3-hydroxybutyrate, the yield is 0.81g/L, and the mass fraction of the polyhydroxyalkanoate accounting for the dry weight of the cells is 32.58%.
Example 8: production of polyhydroxyalkanoate by using glucose and propionic acid as mixed carbon source
Detailed description of the invention according to example 2, except that the culture medium in the experimental group was a liquid TYS medium containing 20g/L glucose and different concentrations of propionic acid (2g/L, 4g/L, 6g/L or 8 g/L); in step 4, the accumulation of poly (3-hydroxybutyrate-co-3-hydroxyvalerate) was detected and calculated as in example 6. The results of the experiment are shown in table 1.
TABLE 1 production of polyhydroxyalkanoates using glucose and propionic acid as mixed carbon sources
When glucose and propionic acid are used as a mixed carbon source, poly (3-hydroxybutyrate-co-3-hydroxyvalerate) can be obtained by fermentation culture, the dry weight of cells is reduced along with the increase of the concentration of the propionic acid, the yield of PHBV and the proportion of the PHBV to the dry weight of the cells are also reduced along with the increase of the concentration of the propionic acid, and the content of 3-hydroxyvalerate monomer is increased along with the increase of the concentration of the propionic acid and is up to more than 72 mol%. When 2g/L of propionic acid is added, the yield of PHBV is highest and is 4.07g/L, wherein the content of 3-hydroxyvaleric acid monomer is 27.28 mol%.
Example 9: production of polyhydroxyalkanoates by using maltose and propionic acid as mixed carbon source
Specific operation according to example 8 of the present invention, except that the medium in the experimental group was a liquid TYS medium containing 20g/L maltose and various concentrations of propionic acid (2g/L, 4g/L, 6g/L, 8 g/L). The results of the experiment are shown in table 2.
TABLE 2 production of polyhydroxyalkanoates using maltose and propionic acid as mixed carbon sources
When maltose and propionic acid are used as a mixed carbon source, poly (3-hydroxybutyrate-co-3-hydroxyvalerate) can be obtained through fermentation culture, the yield and the proportion of the PHBV to the dry weight of cells are also reduced along with the increase of the concentration of the propionic acid, and the content of the 3-hydroxyvalerate monomer is increased along with the increase of the concentration of the propionic acid and is up to more than 95 mol%. The PHBV yield is highest when 2g/L propionic acid is added, the yield is 2.03g/L, and the content of the 3-hydroxyvaleric acid monomer is 59.83 mol%.
Example 10: production of polyhydroxyalkanoate by using soluble starch and propionic acid as mixed carbon source
Detailed description of the invention according to example 8, except that the culture medium in the experimental group was a liquid TYS medium containing 20g/L of soluble starch and different concentrations of propionic acid (2g/L, 4g/L, 6g/L or 8 g/L); in step 4, the accumulation of poly (3-hydroxybutyrate-co-3-hydroxyvalerate) was detected and calculated as in example 6. The results of the experiment are shown in table 3.
TABLE 3 production of polyhydroxyalkanoates using soluble starch and propionic acid as mixed carbon source
When soluble starch and propionic acid are used as a mixed carbon source, poly (3-hydroxybutyrate-co-3-hydroxyvalerate) can be obtained by fermentation culture, and the content of the 3-hydroxyvalerate monomer is increased along with the increase of the concentration of the propionic acid, and the content of the 3-hydroxyvalerate monomer is up to more than 80 mol%. The highest yield of PHBV was obtained by adding 6g/L propionic acid, the yield was 4.81g/L, wherein the content of 3-hydroxyvaleric acid monomer was 79.91 mol%.
Example 11: production of polyhydroxyalkanoate from glucose and natural seawater
Specific operation according to example 2 of the present invention, except that the culture medium in the experimental group was a natural seawater culture medium containing 20g/L glucose.
According to calculation, the culture medium is prepared by utilizing natural seawater and glucose, the dry weight of the cells is 4.92g/L, the polyhydroxy fatty acid ester accumulated in the cells is poly-3-hydroxybutyrate, the yield is 4.44g/L, and the mass fraction of the polyhydroxy fatty acid ester in the dry weight of the cells is 90.27%.
SEQUENCE LISTING
<110> Beijing university of chemical industry
<120> Vibrio costalis for producing polyhydroxyalkanoate and application thereof
<130> GNCLW210003
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 1516
<212> DNA
<213> Salinivibrio costicola
<400> 1
agagtttgat cctggctcag attgaacgct ggcggcaggc ctaacacatg caagtcgagc 60
ggaaacggca gtattgaagc ttcggtggat ttactggacg tcgagcggcg gacgggtgag 120
taacggctgg gaacctgccc tgacgagggg gataaccgtt ggaaacgacg gctaataccg 180
cataatgtct acggaccaaa ggtggcctct acatgtaagc tatcgcgtcg ggatgggccc 240
agttaggatt agctagttgg taaggtaacg gcttaccaag gcgacgatcc ttagctggtt 300
tgagaggatg atcagccaca ctgggactga gacacggccc agactcctac gggaggcagc 360
agtggggaat attgcacaat gggggagacc ctgatgcagc catgccgcgt gtgtgaagaa 420
ggccttcggg ttgtaaagca ctttcagcag tgaggaaggt agtgtactta atacgtgcat 480
tgcttgacgt tagctgcaga agaagcaccg gctaactccg tgccagcagc cgcggtaata 540
cggagggtgc gagcgttaat cggaattact gggcgtaaag cgcatgcagg cggtttgtta 600
agtcagatgt gaaagcccgg ggctcaacct cggaaccgca tttgaaactg gcaggctaga 660
gtcttgtaga ggggggtaga atttcaggtg tagcggtgaa atgcgtagag atctgaagga 720
ataccagtgg cgaaggcggc cccctggaca aagactgacg ctcagatgcg aaagcgtggg 780
tagcaaacag gattagatac cctggtagtc cacgccgtaa acgatgtcta cttggaggct 840
gaggtttaag actttggctt tcggagctaa cgcattaagt agaccgcctg gggagtacgg 900
ccgcaaggtt aaaactcaaa tgaattgacg ggggcccgca caagcggtgg agcatgtggt 960
ttaattcgat gcaacgcgaa gaaccttacc tactcttgac atccagcgaa tcctttagag 1020
atagaggcgt gccttcggga gcgctgagac aggtgctgca tggctgtcgt cagctcgtgt 1080
tgtgaaatgt tgggttaagt cccgcaacga gcgcaaccct tatccttgtt tgccagcacg 1140
taatggtggg aactccaggg agactgccgg tgataaaccg gaggaaggtg gggacgacgt 1200
caagtcatca tggcccttac gagtagggct acacacgtgc tacaatggca gatacagagg 1260
gcagcgagac agcgatgtta agcgaatccc ttaaagtttg tcgtagtccg gattggagtc 1320
tgcaactcga ctccatgaag tcggaatcgc tagtaatcgc agatcagaat gctgcggtga 1380
atacgttccc gggccttgta cacaccgccc gtcacaccat gggagtgggc tgcaccagaa 1440
gtagatagct taaccttcgg gagggcgttt accacggtgt ggttcatgac tggggtgaag 1500
tcgtaacaag gtaacc 1516
Claims (9)
1. Vibrio costigena characterized by: the Vibrio costicola is Salinivibrio costicola, the strain number of the Vibrio costicola is Salinivibrio costicola-TGB-10, and the preservation number of the Vibrio costicola is CGMCC No.21104 in the common microorganism center of China Committee for culture Collection of microorganisms.
2. Use of Vibrio costata according to claim 1 in any one of:
1) producing polyhydroxyalkanoate;
2) preparing a product for producing polyhydroxyalkanoate.
3. Use according to claim 2, characterized in that: the carbon source used for producing the polyhydroxyalkanoate is a saccharide raw material and/or volatile fatty acid.
4. Use according to claim 3, characterized in that: the saccharide raw material is selected from at least one of glucose, maltose and soluble starch;
the volatile fatty acid is selected from at least one of acetic acid, propionic acid and butyric acid.
5. Use according to any one of claims 2-4, characterized in that: the polyhydroxyalkanoate comprises poly-3-hydroxybutyrate and/or poly (3-hydroxybutyrate-co-3-hydroxyvalerate).
6. A method for producing polyhydroxyalkanoate, comprising the steps of: fermentatively culturing the Vibrio costigena of claim 1 with a medium containing the carbon source of claim 3 or 4 to obtain a fermentation product; obtaining the polyhydroxyalkanoate of claim 2 or 5 from the fermentation product.
7. The method of claim 6, wherein: the culture medium comprises an artificial seawater culture medium or a natural seawater culture medium;
the artificial seawater culture medium consists of a solute and a solvent, wherein the solvent is artificial seawater, the concentration of the solute is 1-5 g/L of peptone, 0-5 g/L of yeast powder and 10-30 g/L of a carbon source;
the natural seawater culture medium is composed of a solute and a solvent, the solvent is natural seawater, the solute and the concentration thereof are 5-20 g/L of corn steep liquor, and the carbon source is 10-30 g/L.
8. The method of claim 7, wherein: the artificial seawater culture medium or the natural seawater culture medium also comprises 15g/L agar powder component to prepare a solid culture medium.
9. The method according to claim 7 or 8, characterized in that: when the carbon source is a saccharide raw material or the volatile fatty acid, the obtained polyhydroxyalkanoate is poly-3-hydroxybutyrate;
when the carbon source is a mixture of the saccharide raw material and the propionic acid, the obtained polyhydroxyalkanoate is poly (3-hydroxybutyrate-co-3-hydroxyvalerate).
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110571791.3A CN113265357B (en) | 2021-05-25 | 2021-05-25 | Vibrio costalis for producing polyhydroxyalkanoate and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110571791.3A CN113265357B (en) | 2021-05-25 | 2021-05-25 | Vibrio costalis for producing polyhydroxyalkanoate and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113265357A true CN113265357A (en) | 2021-08-17 |
| CN113265357B CN113265357B (en) | 2022-04-19 |
Family
ID=77232795
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110571791.3A Active CN113265357B (en) | 2021-05-25 | 2021-05-25 | Vibrio costalis for producing polyhydroxyalkanoate and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113265357B (en) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102226206A (en) * | 2011-05-11 | 2011-10-26 | 北京联合大学生物化学工程学院 | Method for preparing polyhydroxybutyrate (PHB) |
| CN103074286A (en) * | 2013-01-28 | 2013-05-01 | 北京大学 | High-salt heterotrophic nitrification-aerobic denitrification dephosphorization salinivibrio and application of salinivibrio in wastewater treatment |
| CN105624075A (en) * | 2016-04-05 | 2016-06-01 | 国家海洋局第一海洋研究所 | Salinivibrio costicola strain |
| CN106119181A (en) * | 2016-06-30 | 2016-11-16 | 江南大学 | A kind of genetic engineering bacterium producing poly butyric hydroxyl valerate and application process thereof |
| CN111206058A (en) * | 2020-03-03 | 2020-05-29 | 北京化工大学 | Method for producing polyhydroxyalkanoate by using acetic acid or butyric acid |
-
2021
- 2021-05-25 CN CN202110571791.3A patent/CN113265357B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102226206A (en) * | 2011-05-11 | 2011-10-26 | 北京联合大学生物化学工程学院 | Method for preparing polyhydroxybutyrate (PHB) |
| CN103074286A (en) * | 2013-01-28 | 2013-05-01 | 北京大学 | High-salt heterotrophic nitrification-aerobic denitrification dephosphorization salinivibrio and application of salinivibrio in wastewater treatment |
| CN105624075A (en) * | 2016-04-05 | 2016-06-01 | 国家海洋局第一海洋研究所 | Salinivibrio costicola strain |
| CN106119181A (en) * | 2016-06-30 | 2016-11-16 | 江南大学 | A kind of genetic engineering bacterium producing poly butyric hydroxyl valerate and application process thereof |
| CN111206058A (en) * | 2020-03-03 | 2020-05-29 | 北京化工大学 | Method for producing polyhydroxyalkanoate by using acetic acid or butyric acid |
Non-Patent Citations (2)
| Title |
|---|
| DOAN VAN THUOC ET AL.: "Utilization of waste fish oil and glycerol as carbon sources for polyhydroxyalkanoate production by Salinivibrio sp. M318", 《INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES》 * |
| 王梦汝 等: "海生杆菌属的基因组测序数据分析", 《生物工程学报》 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN113265357B (en) | 2022-04-19 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Santhanam et al. | Microbial production of polyhydroxy alkanotes (PHA) from Alcaligens spp. and Pseudomonas oleovorans using different carbon sources | |
| Mothes et al. | Production of PHB from crude glycerol | |
| Yu et al. | Conversion of food industrial wastes into bioplastics | |
| Shah | Original research article optimization and production of Polyhydroxybutarate (PHB) by Bacillus subtilis G1S1from soil | |
| Rohini et al. | Synthesis and characterization of poly-beta-hydroxybutyrate from Bacillus thuringiensis R1 | |
| Faccin et al. | Influence of oxygen transfer rate on the accumulation of poly (3-hydroxybutyrate) by Bacillus megaterium | |
| Khiyami et al. | Polyhydroxyalkanoates production via Bacillus plastic composite support (PCS) biofilm and date palm syrup | |
| Lee et al. | Finding of novel lactate utilizing Bacillus sp. YHY22 and its evaluation for polyhydroxybutyrate (PHB) production | |
| CN101363034B (en) | Method for producing polyhydroxyalkanoate using engineering strain | |
| CN111206058B (en) | Method for producing polyhydroxyalkanoate by using acetic acid or butyric acid | |
| CN102226206B (en) | Method for preparing polyhydroxybutyrate (PHB) | |
| Yamane et al. | Polyhydroxyalkanoate synthesis from alcohols during the growth of Paracoccus denitrificans | |
| Singhaboot et al. | A higher in value biopolymer product of polyhydroxyalkanoates (PHAs) synthesized by Alcaligenes latus in batch/repeated batch fermentation processes of sugar cane juice | |
| Kobayashi et al. | Biosynthesis and characterization of poly (3-hydroxybutyrate-co-3-hydroxyhexanoate) from oils and fats by Aeromonas sp. OL-338 and Aeromonas sp. FA-440 | |
| CN113265357B (en) | Vibrio costalis for producing polyhydroxyalkanoate and application thereof | |
| CN112430557B (en) | high-PHA-yield Kabayeri strain, screening method and PHA production method thereof | |
| Khanafari et al. | Production and recovery of poly-β-hydroxybutyrate from whey degradation by Azotobacter | |
| Tabandeh et al. | Biosynthesis of poly-β-hydroxybutyrate as a biodegradable polymer | |
| CN112625962B (en) | Application of sea bacillus in preparation of polyhydroxyalkanoate | |
| CN116574645A (en) | Bacillus cereus MG1 and application thereof | |
| CN116925981A (en) | High-temperature halophilic bacteria and application thereof | |
| CN113088475B (en) | Vibrio salina and application thereof | |
| CN113265356B (en) | Vibrio salina utilizing volatile fatty acid and application thereof | |
| CN102839140B (en) | L-lactic acid producing strain separated and screened out of corn soaking water | |
| CN113832084B (en) | Vibrio alginolyticus for producing polyhydroxyalkanoate and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |