CN113088475B - Vibrio salina and application thereof - Google Patents
Vibrio salina and application thereof Download PDFInfo
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Abstract
The invention discloses vibrio salina and application thereof. The Vibrio salinus is Salinivibrio kushneri, the strain number of the Vibrio salinus is TGB-11, and the preservation number of the Vibrio salinus in the common microorganism center of China Committee for culture Collection of microorganisms is CGMCC No. 21229. The vibrio salinus is applied to any one of the following components: 1) producing polyhydroxyalkanoate; 2) preparing a product for producing polyhydroxyalkanoate. The strain can utilize a saccharide raw material (glucose, maltose, sucrose, soluble starch and the like) as a carbon source or a saccharide raw material and propionic acid as a mixed carbon source to quickly grow and accumulate the poly-3-hydroxybutyrate; the invention can reduce the fermentation cost of the polyhydroxyalkanoate and has great significance for promoting the industrial production, application and development of the polyhydroxyalkanoate.
Description
Technical Field
The invention belongs to the fields of microbiology and fermentation engineering, relates to vibrio salina and application thereof, and particularly relates to vibrio salina for producing polyhydroxyalkanoate and application thereof.
Background
In 1938 Smith et al reported the isolation and identification of Vibrio costicolus. In 1996, Mellado et al reclassified Vibrio costicolus as a representative species of Salinivibrio of the genus Halivibrio based on 16S rRNA gene sequence comparison or the like. Salinivibrio has a certain relationship with Vibrio. The genus halovibrio includes bacteria which are generally gram negative, facultative anaerobic, capable of growing in the NaCl range of 0.5 to 20% (w/v), catalase and oxidase positive, with a genomic G + C content ranging from 49.0 to 51.0 mol% (Salinivibrio kusehner sp. nov., amylolytic halophilic bacteria isolated from microorganisms, Systematic and Applied Microbiology,2018,41, 159. 166. doi:10.1016/j. syapm.2017.12.001). By 2019, species discovered by the genus salinavibrio include s.costicola, s.proteoliticus, s.siamensis, s.sharmensis and s.kushinferi. Wherein S.costicola includes two subspecies, S.costicola subsp.costicola and S.costicola subsp.alcaliphilus (propagation of Salinivibrio socompensis. nov., A new halophilic bacterium isolated from the high-purity hypersaline lake Socomp. Argentina, Microorganisms 2019,7,241, doi: 10.3390/microanginisms 7080241).
Glucose is the most abundant monosaccharide in nature. Glucose, an important carbohydrate, is the most commonly used carbon source in the microbial fermentation industry. Maltose and sucrose are two important common disaccharides. Maltose is formed by condensation of two glucose molecules through alpha, -1,4 glycosidic bonds, and is the main hydrolysate of macromolecular polysaccharide substances such as starch, glycogen, dextrin and the like under the catalysis of beta-amylase. Sucrose, which is formed by dehydration condensation of one molecule of glucose and one molecule of fructose, is widely distributed in plants, is extremely high in sugar beet, sugar cane and fruits, and is the main form of storing, accumulating and transporting sugar for plants. Starch is an important food and industrial raw material, is widely distributed, is low in price, is insoluble in water and common organic solvents, and brings inconvenience to production and application. Soluble starches are prepared by subjecting starch to a mild acid or alkali treatment, the solution of which has good fluidity when exposed to heat.
Polyhydroxyalkanoate (PHA) is a high molecular polymer synthesized in cells during unbalanced growth and metabolism (e.g., excess carbon source and deficiency of nitrogen and phosphorus) of microorganisms, and is used as a carbon source and energy storage substance. PHAs have plastic-like material properties and are completely degraded by microorganisms to water and carbon dioxide in the environment. PHA replaces the traditional non-degradable plastic material from petroleum, is helpful to solve the increasingly serious problem of white garbage pollution, and has good application prospect.
Disclosure of Invention
The invention aims to provide vibrio salina and application thereof, and particularly provides vibrio salina for producing polyhydroxyalkanoate and application thereof.
The vibrio salinus provided by the invention is Salinivibrio kushneri, the strain number of the vibrio salinus is TGB-11, and the preservation number of the vibrio salinus in the common microorganism center of China Committee for culture Collection of microorganisms is CGMCC No. 21229.
The Vibrio salinus is Salinivibrio kushni TGB-11, which is called S.kushni TGB-11 for short.
The vibrio salinus is applied to any one of the following components:
1) producing polyhydroxyalkanoate;
2) preparing a product for producing polyhydroxyalkanoate.
In the application of the vibrio salini, the carbon source used for producing the polyhydroxyalkanoate is a carbohydrate raw material.
In the application of the vibrio salini, the carbon source used for producing the polyhydroxyalkanoate is volatile fatty acid.
In the application of the vibrio salini, the carbon source used for producing the polyhydroxyalkanoate is a carbohydrate raw material and volatile fatty acid.
In the above application of Vibrio salina, the saccharide raw material is at least one selected from glucose, maltose, soluble starch and sucrose.
In the above application of vibrio salinus, the volatile fatty acid comprises propionic acid.
In the application of the vibrio salina, the polyhydroxyalkanoate comprises poly-3-hydroxybutyrate (PHB for short) and/or poly (3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV for short). 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 salina 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 10g/L of ammonium sulfate, 10g/L of dipotassium hydrogen phosphate, and 10-30 g/L of a carbon source.
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 invention, the artificial seawater culture medium is a liquid culture medium which is prepared from the following components in concentration: 5g/L of peptone and 1g/L of yeast powder, wherein the solvent of the artificial seawater culture medium is artificial seawater.
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, sucrose, 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 78 percent at most, and the yield in a shake flask can reach 5 g/L.
2. The strain can rapidly grow and accumulate poly (3-hydroxybutyrate-co-3-hydroxyvalerate) by using sugar raw materials (glucose, maltose, sucrose, soluble starch and the like) and propionic acid (the concentration is 0.5g/L-2g/L) as a mixed carbon source, the proportion of polyester to the dry weight of bacteria can reach 50 percent at most, the yield in a shake flask can reach 4g/L, and the content of 3HV monomers can reach 40 percent at most.
3. The invention can reduce the fermentation cost of the polyhydroxyalkanoate and has great significance for promoting the industrial production, application and development of the polyhydroxyalkanoate.
Deposit description
Classification nomenclature of biological materials: vibrio salina.
Latin name of biomaterial: salinivibrio kushneri.
According to the biological materials (strains): TGB-11.
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: 2020.11.25.
the preservation number is: CGMCC No. 21229.
Drawings
FIG. 1 is a TEM photograph of S.kushneri TGB-11 strain in TYS medium supplemented with glucose as a carbon source in example 1 of the present invention.
FIG. 2 is a TEM photograph of S.kushneri TGB-11 strain in TYS medium in 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 ═ cell dry weight (PHV peak area in sample/internal standard peak area in sample) × [ (internal standard peak area/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.
The composition of TYS broth containing 20g/L glucose was as follows: 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 20g/L of glucose.
The composition of the liquid TYS medium containing 20g/L maltose was as follows: 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 20g/L of maltose.
The composition of the liquid TYS medium containing 20g/L of soluble starch was as follows: the artificial seawater desalination solution is a solution with the pH of 7.5 and composed of 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 20g/L of soluble starch.
The composition of the liquid TYS medium containing 20g/L of sucrose was as follows: 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 20g/L of sucrose.
The composition of the liquid TYS medium containing 20g/L of acetic acid was as follows: 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 20g/L of acetic acid.
The composition of the liquid TYS medium containing 10g/L of butyric acid was as follows: 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.
The composition of the liquid TYS medium containing 20g/L of glucose and different concentrations of propionic acid (0.5g/L, 1g/L, 1.5g/L or 2g/L) was as follows: 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 glucose, and 0.5g/L, 1g/L, 1.5g/L or 2g/L of propionic acid.
The composition of the liquid TYS medium containing 1.5g/L of propionic acid and 20g/L of a different disaccharide (sucrose, maltose or soluble starch) is as follows: 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, 1.5g/L of propionic acid and 20g/L of disaccharide.
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 10g/L of ammonium sulfate, 10g/L of dipotassium hydrogen phosphate and 20g/L of glucose.
Example 1 isolation and characterization of Salinivibrio kushni TGB-11
1. Isolation of Salinivibrio kushni TGB-11
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. Wherein one strain of separated and purified TGB-11 is Salinivibrio kushni TGB-11.
2. Identification of Salinivibrio kushni TGB-11
(1) Observation of culture characteristics of strain TGB-11
The strain TGB-11 is inoculated on a TYS culture medium and cultured for 12h at 30 ℃ to form a round colony with the diameter of 1.2-1.8mm, smooth edge, convex surface and milky aggregation. The strain TGB-11 cells were rod-shaped, oblong and slightly bent as observed by transmission electron microscopy.
(2) Analysis of physiological and biochemical characteristics
The strain TGB-11 is gram-negative bacteria, can grow on TYS and LB culture medium, 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 strain TGB-11 was as follows:
AGAGTTTGATCCTGGCTCAGATTGAACGCTGGCGGCAGGCCTAACACATGCAAGTCGAGCGGAAACGGCAGCATTGAAGCTTCGGTGGATTTGCTGGACGTCGAGCGGCGGACGGGTGAGTAACGGCTGGGAACCTGCCCTGACGAGGGGGATAACCGTTGGAAACGACGGCTAATACCGCATAATGTCTTAGTTCATTACGAGCTGGGACCAAAGGTGGCCTCTACATGTAAGCTATCGCGTTGGGATGGGCCCAGTTAGGATTAGCTAGTTGGTAAGGTAATGGCTTACCAAGGCAACGATCCTTAGCTGGTTTGAGAGGATGATCAGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGGGAGACCCTGATGCAGCCATGCCGCGTGTGTGAAGAAGGCCTTCGGGTTGTAAAGCACTTTCAGCAGTGAGGAAGGTGGTGTACTTAATACGTGCATTGCTTGACGTTAGCTGCAGAAGAAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCGAGCGTTAATCGGAATTACTGGGCGTAAAGCGCATGCAGGCGGTTTGTTAAGTCAGATGTGAAAGCCCGGGGCTCAACCTCGGAACCGCATTTGAAACTGGCAGGCTAGAGTCTTGTAGAGGGGGGTAGAATTTCAGGTGTAGCGGTGAAATGCGTAGAGATCTGAAGGAATACCAGTGGCGAAGGCGGCCCCCTGGACAAAGACTGACGCTCAGATGCGAAAGCGTGGGTAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGCTGTCTACTTGGAGGTTGAGGTTTAAGACTTTGGCTTTCGGCGCTAACGCATTAAGTAGACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTACTCTTGACATCCAGAGAACTTTCCAGAGATGGATTGGTGCCTTCGGGAACTCTGAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTTGTGAAATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATCCTTGTTTGCCAGCACGTAATGGTGGGAACTCCAGGGAGACTGCCGGTGATAAACCGGAGGAAGGTGGGGACGACGTCAAGTCATCATGGCCCTTACGAGTAGGGCTACACACGTGCTACAATGGCAGATACAGAGGGCAGCGAAGCTGCGAAGTGGAGCGAATCCCTTAAAGTCTGTCGTAGTCCGGATTGGAGTCTGCAACTCGACTCCATGAAGTCGGAATCGCTAGTAATCGTGGATCAGAATGCCACGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTGGGCTGCACCAGAAGTAGATAGCTTAACCTTCGGGAGGGCGTTTACCACGGTGTGGTTCATGACTGGGGTGAAGTCGTAACAAGGTAACC
the 16S rDNA gene sequence of the strain TGB-11 is 1531bp, and the gene sequence comparison analysis shows that the similarity of the gene sequences of the strain TGB-11 and the strain in Salinivibrio is higher, and the strain with the highest similarity is S.kushni AL184 (99.68%).
The strain TGB-11 of the present invention was finally determined to be Vibrio salina (Salinivibrio kushinhei) by combining the above morphological, physiological and biochemical characteristics, and the results of the genomic sequence determination.
The strain GTB-11 has been preserved in 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, 25. The preservation number is CGMCC No. 21229. The strain name: vibrio salina; latin name: salinivibrio kushni; according to the biological materials (strains): TGB-11.
Example 2: production of polyhydroxyalkanoates using glucose as carbon source
1. Preparation of seed liquid of Vibrio salina S.kushneri TGB-11 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 containing no glucose 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 in a total liquid volume of 40mL to prepare a fermentation liquid 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;
the polymer was quantitatively analyzed by an internal standard method using a 1. mu.L microsyringe from Agilent, and the amount was determined on the basis of 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 6.35g/L, the polyhydroxy fatty acid ester accumulated in the cell is poly-3-hydroxybutyrate, the yield is 5.00g/L, and the mass fraction of the polyhydroxy fatty acid ester in the dry weight of the cell is 78.71%.
In the control group, when glucose was not added, the dry cell weight was 1.44g/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 substrate
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 maltose.
By calculation, the dry weight of the cells when maltose is used as a carbon source is 5.82g/L, the polyhydroxyalkanoate ester accumulated in the cells is poly-3-hydroxybutyrate, the yield is 3.73g/L, and the mass fraction of the polyhydroxyalkanoate ester accounting for the dry weight of the cells is 64.16%.
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 6.78g/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 48.36% of the dry weight of the cells.
Example 5: production of polyhydroxyalkanoate by using sucrose as carbon source
Specific operation according to example 2 of the present invention, except that the culture medium in the experimental group was a liquid TYS medium containing 20g/L sucrose.
By calculation, the dry weight of the cells when sucrose is used as a carbon source is 5.62g/L, the polyhydroxyalkanoate ester accumulated in the cells is poly-3-hydroxybutyrate, the yield is 3.23g/L, and the mass fraction of the polyhydroxyalkanoate ester accounting for the dry weight of the cells is 57.46%.
Example 6: 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 3.90g/L, the polyhydroxyalkanoate accumulated in the cells is poly-3-hydroxybutyrate, the yield is 0.77g/L, and the mass fraction of the polyhydroxyalkanoate accounting for the dry weight of the cells is 19.72%.
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 2.90g/L, the polyhydroxyalkanoate accumulated in the cells is poly-3-hydroxybutyrate, the yield is 0.88g/L, and the mass fraction of the polyhydroxyalkanoate accounts for 30.34% of the dry weight of the cells.
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 (0.5g/L, 1g/L, 1.5g/L or 2 g/L); and 4, detecting the accumulation of the poly (3-hydroxybutyrate-co-3-hydroxyvalerate) in the step 4 and calculating. 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 propionic acid, the yield of PHBV and the proportion of PHBV to the dry weight of cells are also reduced along with the increase of the concentration of propionic acid, and the content of 3-hydroxyvalerate monomer is increased along with the increase of the concentration of propionic acid and is maximum over 42 mol%. The highest yield of PHBV is obtained when 0.5g/L propionic acid is added, the yield is 4.38g/L, and the content of the 3-hydroxyvaleric acid monomer is 8.57 mol%.
Example 9: production of polyhydroxyalkanoates by using different disaccharides 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 1.5g/L propionic acid and 20g/L of different disaccharides (sucrose, maltose or soluble starch); and 4, detecting the accumulation of the poly (3-hydroxybutyrate-co-3-hydroxyvalerate) in the step 4 and calculating. The results of the experiment are shown in table 2.
TABLE 2 production of polyhydroxyalkanoates using different disaccharides and propionic acid as mixed carbon sources
The poly (3-hydroxybutyrate-co-3-hydroxyvalerate) can be obtained by fermentation culture with sucrose, maltose and soluble starch added with 1.5g/L of propionic acid as mixed carbon sources. The dry weight of the cells is more than 7g/L, and the content of the 3-hydroxyvaleric acid monomer is 17.40mol percent to 30.00mol percent. Of the three different disaccharides, sucrose was most effective and PHBV yield was 4.62 g/L. Considering that sucrose is a raw material with wide sources, the strain provided by the invention has good application prospect in the aspect of producing PHBV by utilizing sucrose and propionic acid.
Example 10: production of polyhydroxyalkanoate from glucose and seawater
Specifically, according to example 2 of the present invention, the culture medium in the experimental group was a natural seawater culture medium containing 20g/L glucose (ammonium sulfate 10g/L, dipotassium hydrogen phosphate 10g/L, solvent of the natural seawater culture medium was seawater obtained from the offshore area of coastal new area of Tianjin).
By calculation, the dry weight of the cells when the glucose and the seawater are used as raw materials is 3.03g/L, the polyhydroxy fatty acid ester accumulated in the cells is poly-3-hydroxybutyrate, the yield is 2.64g/L, and the mass fraction of the polyhydroxy fatty acid ester in the dry weight of the cells is 87.13%.
SEQUENCE LISTING
<110> Beijing university of chemical industry
<120> Vibrio salina and application thereof
<130> GNCLW210004
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 1531
<212> DNA
<213> Salinivibrio kushneri
<400> 1
agagtttgat cctggctcag attgaacgct ggcggcaggc ctaacacatg caagtcgagc 60
ggaaacggca gcattgaagc ttcggtggat ttgctggacg tcgagcggcg gacgggtgag 120
taacggctgg gaacctgccc tgacgagggg gataaccgtt ggaaacgacg gctaataccg 180
cataatgtct tagttcatta cgagctggga ccaaaggtgg cctctacatg taagctatcg 240
cgttgggatg ggcccagtta ggattagcta gttggtaagg taatggctta ccaaggcaac 300
gatccttagc tggtttgaga ggatgatcag ccacactggg actgagacac ggcccagact 360
cctacgggag gcagcagtgg ggaatattgc acaatggggg agaccctgat gcagccatgc 420
cgcgtgtgtg aagaaggcct tcgggttgta aagcactttc agcagtgagg aaggtggtgt 480
acttaatacg tgcattgctt gacgttagct gcagaagaag caccggctaa ctccgtgcca 540
gcagccgcgg taatacggag ggtgcgagcg ttaatcggaa ttactgggcg taaagcgcat 600
gcaggcggtt tgttaagtca gatgtgaaag cccggggctc aacctcggaa ccgcatttga 660
aactggcagg ctagagtctt gtagaggggg gtagaatttc aggtgtagcg gtgaaatgcg 720
tagagatctg aaggaatacc agtggcgaag gcggccccct ggacaaagac tgacgctcag 780
atgcgaaagc gtgggtagca aacaggatta gataccctgg tagtccacgc cgtaaacgct 840
gtctacttgg aggttgaggt ttaagacttt ggctttcggc gctaacgcat taagtagacc 900
gcctggggag tacggccgca aggttaaaac tcaaatgaat tgacgggggc ccgcacaagc 960
ggtggagcat gtggtttaat tcgatgcaac gcgaagaacc ttacctactc ttgacatcca 1020
gagaactttc cagagatgga ttggtgcctt cgggaactct gagacaggtg ctgcatggct 1080
gtcgtcagct cgtgttgtga aatgttgggt taagtcccgc aacgagcgca acccttatcc 1140
ttgtttgcca gcacgtaatg gtgggaactc cagggagact gccggtgata aaccggagga 1200
aggtggggac gacgtcaagt catcatggcc cttacgagta gggctacaca cgtgctacaa 1260
tggcagatac agagggcagc gaagctgcga agtggagcga atcccttaaa gtctgtcgta 1320
gtccggattg gagtctgcaa ctcgactcca tgaagtcgga atcgctagta atcgtggatc 1380
agaatgccac ggtgaatacg ttcccgggcc ttgtacacac cgcccgtcac accatgggag 1440
tgggctgcac cagaagtaga tagcttaacc ttcgggaggg cgtttaccac ggtgtggttc 1500
atgactgggg tgaagtcgta acaaggtaac c 1531
Claims (8)
1. Vibrio salina, characterized by: the Vibrio salinus is Salinivibrio kushneri, the strain number of the Vibrio salinus is TGB-11, and the preservation number of the Vibrio salinus in the common microorganism center of China Committee for culture Collection of microorganisms is CGMCC No. 21229.
2. The use of Vibrio salina of claim 1 in any one of:
1) producing polyhydroxyalkanoate;
2) preparing a product for the production of polyhydroxyalkanoate;
the polyhydroxyalkanoate is poly-3-hydroxybutyrate and/or poly (3-hydroxybutyrate-co-3-hydroxyvalerate).
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 comprises propionic acid.
5. A method for producing polyhydroxyalkanoate, comprising the steps of: fermentatively culturing the Vibrio salini of claim 1 in a medium containing the carbon source of claim 3 or 4 to obtain a fermentation product; obtaining polyhydroxyalkanoate from the fermentation product;
the polyhydroxyalkanoate is poly-3-hydroxybutyrate and/or poly (3-hydroxybutyrate-co-3-hydroxyvalerate).
6. The method of claim 5, 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 10g/L of ammonium sulfate, 10g/L of dipotassium hydrogen phosphate, and 10-30 g/L of a carbon source.
7. The method of claim 6, 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.
8. The method according to claim 6 or 7, characterized in that: when the carbon source is a saccharide raw material, the obtained polyhydroxyalkanoate is poly-3-hydroxybutyrate;
when the carbon source is a mixture of the saccharide raw material and propionic acid, the obtained polyhydroxyalkanoate is poly (3-hydroxybutyrate-co-3-hydroxyvalerate).
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| WO2012165131A1 (en) * | 2011-05-27 | 2012-12-06 | 独立行政法人理化学研究所 | Novel marine microorganism, and method for producing polyhydroxyalkanoate |
| CN110857449A (en) * | 2018-08-24 | 2020-03-03 | 清华大学 | Improved method for producing polyhydroxyalkanoate |
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