CN113088475B - A kind of salt vibrio and its application - Google Patents

Abstract

The invention discloses a salt vibrio and its application. The salt vibrio of the present invention is Salinivibrio kushneri, its strain number is TGB-11, and its preservation number in the General Microbiology Center of the China Microorganism Culture Collection Management Committee is CGMCC No. 21229. The application of the salt vibrio of the present invention in any one of the following: 1) production of polyhydroxyalkanoate; 2) preparation of a product for producing polyhydroxyalkanoate. The strain of the present invention can use carbohydrate raw materials (glucose, maltose, sucrose, soluble starch, etc.) as carbon sources or carbohydrate raw materials and propionic acid as mixed carbon sources to rapidly grow and accumulate poly-3-hydroxybutyrate; the present invention can reduce The fermentation cost of polyhydroxyalkanoates is of great significance for promoting the industrial production and application development of polyhydroxyalkanoates.

Description

A kind of salt vibrio and its application

technical field

The invention belongs to the field of microbiology and fermentation engineering, and relates to a salvibrio bacterium and an application thereof, in particular to a salvibrio bacterium for producing polyhydroxy fatty acid ester and an application thereof.

Background technique

In 1938, Smith et al reported the isolation and identification of Vibrio costicolus. In 1996, Mellado et al. reclassified Vibrio costicolus as Salinivibrio costicola based on the comparison of 16S rRNA gene sequences, as a representative species of Salinivibrio. The genus Salinivibrio has a certain genetic relationship with the genus Vibrio. The bacteria included in the genus Halovibrio are generally gram-negative, facultatively anaerobic, capable of growing in the range of 0.5 to 20% (w/v) NaCl, positive for catalase and oxidase, genome G+C The content ranged from 49.0 to 51.0 mol% (Salinivibrio kushneri sp.nov., amoderately halophilic bacterium isolated fromsalterns, Systematic and Applied Microbiology, 2018, 41, 159-166, doi: 10.1016/j.syapm.2017.12.001). As of 2019, the species found in the Salinivibrio genus include S.costicola, S.proteolyticus, S.siamensis, S.sharmensis and S.kushneri. Among them, S.costicola includes two subspecies, S.costicola subsp.costicola and S.costicola subsp.alcaliphilus (Characterization of Salinivibrio socompensis sp.nov., A new halophilicbacterium isolated from the high-altitude hypersaline lake Socompa, Argentina, Microorganisms 2019 , 7,241, doi:10.3390/microorganisms7080241).

Glucose is the most abundant monosaccharide in nature. As an important carbohydrate, glucose is the most commonly used carbon source in the microbial fermentation industry. Maltose and sucrose are two important common disaccharides. Maltose is formed by the condensation of two glucose molecules through α,-1,4 glycosidic bonds, and is the main hydrolysis product of macromolecular polysaccharides such as starch, glycogen, and dextrin under the catalysis of β-amylase. Sucrose is formed by the dehydration condensation of a molecule of glucose and a molecule of fructose. It is widely distributed in plants and is extremely high in sugar beets, sugar cane and fruits. It is the main form of plant storage, accumulation and transportation of sugar. Starch is an important food and industrial raw material, widely distributed, cheap, insoluble in water, insoluble in common organic solvents, which brings a lot of inconvenience to production and application. Soluble starch is prepared by treating starch with mild acid or alkali, and its solution has good fluidity when heated.

Polyhydroxyalkanoate (PHA) is a macromolecular polymer synthesized in the cell as a carbon source and energy storage material when microorganisms grow and metabolize unbalanced (such as excess carbon source and lack of nitrogen and phosphorus). PHA has plastic-like material properties and can be completely degraded into water and carbon dioxide by microorganisms in the environment. PHA replaces traditional petroleum-derived non-degradable plastic materials, helps to solve the increasingly serious problem of white garbage pollution, and has good application prospects.

SUMMARY OF THE INVENTION

The object of the present invention is to provide a kind of Salovibrio and its application, specifically a kind of Salovibrio for producing polyhydroxy fatty acid ester and its application.

The Salinivibrio kushneri provided by the present invention is Salinivibrio kushneri, its strain number is TGB-11, and its preservation number in the General Microbiology Center of China Microorganism Culture Collection Management Committee is CGMCC No.21229.

The Salinivibrio kushneri TGB-11 in the present invention is referred to as S.kushneri TGB-11 for short.

The application of salt vibrio of the present invention in any of the following:

1) production of polyhydroxyalkanoates;

2) Preparation of products for the production of polyhydroxyalkanoates.

In the application of the above-mentioned Salovibrio, the carbon source used in the production of polyhydroxyalkanoate is a carbohydrate raw material.

In the application of the above-mentioned salt vibrio, the carbon source used in the production of polyhydroxyalkanoate is volatile fatty acid.

In the application of the above-mentioned Salovibrio, the carbon sources used in the production of polyhydroxyalkanoate are carbohydrate raw materials and volatile fatty acids.

In the application of the above-mentioned Halovibrio, the carbohydrate raw material is selected from at least one of glucose, maltose, soluble starch and sucrose.

In the application of the above-mentioned Salovibrio, the volatile fatty acid includes propionic acid.

In the application of the above-mentioned salvibracteria, the polyhydroxyalkanoate comprises poly-3-hydroxybutyrate (PHB for short) and/or poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (referred to as PHBV). The poly(3-hydroxybutyrate-co-3-hydroxyvalerate) is a copolyester containing two monomer components, 3-hydroxybutyric acid and 3-hydroxyvaleric acid.

The present invention further provides a method for producing polyhydroxyalkanoates, comprising the steps of: fermenting and culturing the above-mentioned halovibrio with a medium containing the above-mentioned carbon source to obtain a fermentation product; and obtaining the above-mentioned fermented product from the fermentation product. Polyhydroxyalkanoates.

In the above-mentioned method, the substratum comprises artificial seawater substratum or natural seawater substratum,

The artificial seawater culture medium is composed of a solute and a solvent, the solvent is artificial seawater, the solute and its concentration are peptone 1-5 g/L, yeast powder 0-5 g/L, and carbon source 10-30 g/L ;

The natural seawater culture medium consists of a solute and a solvent, the solvent is natural seawater, the solute and its concentration are 10 g/L of ammonium sulfate, 10 g/L of dipotassium hydrogen phosphate, and 10 to 30 g/L of the carbon source.

In the present invention, the artificial seawater is composed of components including the following concentrations: sodium chloride 27.5g/L, potassium chloride 0.7g/L, magnesium chloride 2.5g/L, magnesium sulfate 3.3g/L, calcium chloride 1g /L, sodium bicarbonate 0.2g/L, the solvent of described artificial seawater is water;

The natural seawater is specifically taken from the coastal waters of Tianjin Binhai New Area.

In the present invention, the artificial seawater culture medium is specifically a liquid culture medium made of the following concentration components: peptone 5g/L, yeast powder 1g/L, and 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 15 g/L of agar powder components 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 the mixture of the carbohydrate raw material and the propionic acid, the obtained polyhydroxyalkanoate is poly(3-hydroxybutyrate-co-3-hydroxypentane) acid ester).

The present invention further provides a culture medium, which is the above-mentioned artificial seawater culture medium or natural seawater culture medium.

The present invention has the following advantages:

1. The bacterial strain of the present invention can utilize carbohydrate raw materials (glucose, maltose, sucrose, soluble starch, etc.) as carbon source to grow rapidly and accumulate poly-3-hydroxybutyrate, and polyester accounts for the highest proportion of bacterial dry weight. up to 78%, and the yield in shake flasks can reach 5g/L.

2. The strain of the present invention can utilize carbohydrate raw materials (glucose, maltose, sucrose, soluble starch, etc.) and propionic acid (concentration 0.5g/L-2g/L) as a mixed carbon source to grow rapidly and accumulate poly(3-hydroxyl) Butyrate-co-3-hydroxyvalerate), polyester accounts for up to 50% of the dry weight of bacteria, the yield in shake flasks can reach 4 g/L, and the 3HV monomer content is up to 40%.

3. The present invention can reduce the fermentation cost of the polyhydroxyalkanoate, and has great significance for promoting the industrial production and application development of the polyhydroxyalkanoate.

Preservation Instructions

Taxonomic nomenclature of biological materials: Salovibrio.

Latin name for biological material: Salinivibrio kushneri.

Participating biological material (strain): TGB-11.

The full name of the preservation unit: General Microbiology Center of China Microorganism Culture Collection Administration Committee.

Abbreviation of the depositary unit: CGMCC.

Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

Deposit date: 2020.11.25.

Deposit number: CGMCC No.21229.

Description of drawings

Fig. 1 is a transmission electron microscope photograph of S. kushneri TGB-11 strain in Example 1 of the present invention in a TYS medium supplemented with glucose as a carbon source.

FIG. 2 is a transmission electron microscope photograph of S. kushneri TGB-11 strain in TYS medium in Example 1 of the present invention.

Detailed ways

The experimental methods used in the following examples are conventional methods unless otherwise specified.

The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.

The quantitative experiments in the following examples are all set to repeat the experiments three times, and the results are averaged. The standard polyhydroxyalkanoate was purchased from Sigma-Aldrich, the product number is 403121, the product name is poly(3-hydroxybutyrate-co-3-hydroxyvalerate), and the content of 3-hydroxybutyric acid monomer is 88mol %, and the 3-hydroxyvaleric acid monomer content was 12 mol%.

The method of freeze-drying in the following examples is as follows:

After fermenting and culturing the microorganisms, take the fermentation broth, centrifuge at 10,000 rpm for 10 minutes, discard the supernatant, resuspend the bacteria with deionized water for washing, and then centrifuge again at 10,000 rpm for 10 minutes to collect the bacteria. Freeze at -20°C for 2 hours, and then freeze-dried in a freeze-vacuum dryer for 10 hours to obtain a freeze-dried product.

The dry weight of cells in the following examples is calculated as the dry weight of cells per liter of fermentation broth. The unit of cell dry weight is g/L. Cell dry weight (CDW for short)=(weight of centrifuge tube after freeze-drying - weight of original empty centrifuge tube)/amount of fermentation broth; weight of centrifuge tube after freeze-drying and original empty centrifuge tube The unit of weight is g; the unit of fermentation broth is L.

The detection method of thalline polyhydroxyalkanoate content in the following embodiment: freeze-dried product carries out esterification reaction, then calculates by measuring the content of product after esterification reaction;

Esterification reaction: take 30-40 mg of freeze-dried product in an esterification tube, add 2 mL of chloroform and 2 mL of esterification solution (the esterification solution is obtained by adding 15 mL of concentrated sulfuric acid and 0.5 g of benzoic acid to 500 mL of methanol) and mix well, Covered and sealed, esterified at 100 °C for 4 hours; after cooling to room temperature, 1 mL of deionized water was added, fully shaken and mixed with a vortex shaker, and left to stand for stratification; after the chloroform phase was completely separated from the water, take 1 μL of the chloroform phase Perform gas chromatographic analysis.

About 20 mg of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) was taken and subjected to esterification in the same manner as a standard product.

Gas chromatographic analysis parameters: HP 6890 gas chromatograph was used, the chromatographic column was HP-5 capillary column, the column length was 30 m, the inner diameter was 320 μm, and the stationary phase was phenylmethyl polysiloxane with a thickness of 25 nm; the detector was flame ionization Detector (Flameionization detector, FID); high-purity nitrogen is used as carrier gas, hydrogen is used as fuel gas, and air is used as auxiliary gas;

The conditions for gas chromatographic analysis are as follows:

(1) Column temperature: start at 80°C and stay for 1.5min; ramp up to 140°C at a rate of 30°C/min and stay for 0min; ramp up to 220°C at a rate of 40°C/min and stay for 1min. The total time is 6.5min.

(2) Column pressure: start at 10 psi, stay for 1.5 min; increase the pressure at a rate of 2.5 psi/min to 20 psi, stay for 0.5 min. (psi is the unit of pressure, ie pounds per square inch, 1psi=6.89476kPa)

(3) Injection port: the temperature is 200°C, the split mode is used, and the split ratio is 30.

(4) Detector: the temperature is 220°C, the hydrogen flow rate is 30mL/min, and the air flow rate is 400mL/min.

Using Agilent's micro-injector, the injection volume was 1 μL, and the internal standard method was used to quantitatively analyze the polymer, which was quantified according to the peak area.

For gas chromatography, lyophilized cell samples were compared to poly(3-hydroxybutyrate-co-3-hydroxyvalerate) standards. The above steps were used for esterification reaction and gas chromatography detection. The freeze-dried cell samples contained the same signal as the 3-hydroxybutyric acid in the standard product, which indicated that the polyhydroxyalkanoate accumulated in the cells was poly-hydroxybutyric acid. 3-hydroxybutyrate. The freeze-dried cell sample contains the same signal as 3-hydroxybutyric acid and 3-hydroxyvaleric acid in the standard product, which means that the polyhydroxyalkanoate accumulated in the bacteria is poly(3-hydroxybutyrate-co) -3-hydroxyvalerate).

The poly-3-hydroxybutyrate yield algorithm is: PHB yield = (PHB peak area in the sample/internal standard peak area in the sample) × [(internal standard peak area in the standard product/PHB peak area in the standard product) × (standard product peak area product mass×0.866)]/sample esterification mass×cell dry weight

The poly(3-hydroxybutyrate-co-3-hydroxyvalerate) yield algorithm is: PHBV yield=PHB yield+PHV yield, where:

PHB yield = (PHB peak area in sample/internal standard peak area in sample) × [(internal standard peak area in standard product/PHB peak area in standard product) × (standard product mass × 0.866)]/sample esterification mass × dry cell weight

PHV yield=(PHV peak area in sample/internal standard peak area in sample)×[(internal standard peak area in standard product/PHV peak area in standard product)×(standard product mass×0.134)]/sample esterification mass× dry cell weight

The polymer content was defined as the ratio of polymer to dry cell weight, polymer content=polymer yield/cell dry weight×100%.

The composition of TYS liquid medium is as follows: it consists of solute and solvent, pH 7.5 solution, the solvent is artificial seawater, the solute and its concentration are peptone 5g/L, and yeast powder 1g/L. Artificial seawater is composed of solute and solvent, the solvent is water, the solute and its concentration are: sodium chloride 27.5g/L, potassium chloride 0.7g/L, magnesium chloride 2.5g/L, magnesium sulfate 3.3g/L, calcium chloride 1g/L, sodium bicarbonate 0.2g/L.

The composition of the TYS liquid medium containing 20g/L glucose is as follows: it consists of a solute and a solvent, the pH is 7.5, the solvent is the above-mentioned artificial seawater, the solute and its concentration are peptone 5g/L, yeast powder 1g/L, glucose 20g /L.

The composition of the liquid TYS medium containing 20g/L maltose is as follows: it consists of a solute and a solvent, a solution with a pH of 7.5, the solvent is the above-mentioned artificial seawater, the solute and its concentration are peptone 5g/L, yeast powder 1g/L, maltose 20g /L.

The composition of the liquid TYS medium containing 20g/L soluble starch is as follows: it consists of a solute and a solvent, a solution with a pH of 7.5, the solvent is the above artificial seawater, the solute and its concentration are peptone 5g/L, yeast powder 1g/L, soluble Starch 20g/L.

The composition of the liquid TYS medium containing 20g/L sucrose is as follows: it consists of a solute and a solvent, a solution with a pH of 7.5, the solvent is the above artificial seawater, the solute and its concentration are peptone 5g/L, yeast powder 1g/L, sucrose 20g /L.

The composition of the liquid TYS medium containing 20g/L acetic acid is as follows: a solution with a pH of 7.5 consisting of a solute and a solvent, the solvent is the above artificial seawater, the solute and its concentration are peptone 5g/L, yeast powder 1g/L, acetic acid 20g /L.

The composition of the liquid TYS medium containing 10g/L butyric acid is as follows: it consists of a solute and a solvent, a solution with a pH of 7.5, the solvent is the above-mentioned artificial seawater, the solute and its concentration are peptone 5g/L, yeast powder 1g/L, Acid 10g/L.

The composition of liquid TYS medium containing 20 g/L glucose and different concentrations of propionic acid (0.5 g/L, 1 g/L, 1.5 g/L or 2 g/L) was as follows: a solution consisting of solute and solvent, pH 7.5, The solvent is the above artificial seawater, the solute and its concentration are peptone 5g/L, yeast powder 1g/L, glucose 20g/L, propionic acid 0.5g/L, 1g/L, 1.5g/L or 2g/L.

The composition of liquid TYS medium of liquid TYS medium containing 1.5g/L propionic acid and 20g/L of different disaccharides (sucrose, maltose or soluble starch) is as follows: a solution consisting of solute and solvent, pH 7.5, solvent is In the above artificial seawater, the solute and its concentration are peptone 5g/L, yeast powder 1g/L, propionic acid 1.5g/L, and disaccharide 20g/L.

The composition of TYS solid medium is as follows: it consists of solute and solvent, the solvent is the above artificial seawater, the solute and its concentration are peptone 5g/L, yeast powder 1g/L, and agar 15g/L.

The composition of the natural seawater culture medium containing 20g/L glucose is as follows: it is composed of a solute and a solvent, the solvent is natural seawater, specifically taken from the coastal waters of Binhai New District, Tianjin, the solute and its concentration are ammonium sulfate 10g/L, dipotassium hydrogen phosphate 10g /L, glucose 20g/L.

Example 1. Separation and identification of Salinivibrio kushneri TGB-11

1. Isolation of Salinivibrio kushneri TGB-11

Water samples were collected from Yanchi in Yanchang, Binhai New Area, Tianjin. Draw 1 mL of water sample and add it to TYS liquid medium containing 20 g/L acetic acid, incubate at 30°C and 200 rpm for 24 h, streak the TYS solid medium containing 20 g/L acetic acid with a sterilized inoculation loop, and invert for 24 h. Pick a single clone, purify it on a TYS solid medium plate, and after it is determined to be pure bacteria, transfer it to a slant at 4 °C for short-term storage, transfer it to a 25% glycerol tube, and store it at -80 °C for a long time. One of the isolated and purified strains TGB-11 is Salinivibrio kushneri TGB-11.

2. Identification of Salinivibrio kushneri TGB-11

(1) Observation of culture characters of strain TGB-11

After the strain TGB-11 was inoculated on TYS medium and cultivated at 30°C for 12 hours, it formed round colonies of 1.2-1.8 mm, with smooth edges and raised surfaces, and aggregated into milky white round colonies. Transmission electron microscopy showed that the cells of strain TGB-11 were rod-shaped, oblong and slightly curved.

(2) Analysis of physiological and biochemical characteristics

Strain TGB-11 is a Gram-negative bacteria that can grow on TYS and LB medium, and the optimum medium is TYS medium; the growth temperature is 20-45°C, and the optimum temperature is 30°C; the pH range for growth is 6-10, the optimum pH is 7; the range of sodium chloride for growth is 1-20% (w/v), and the optimum concentration of sodium chloride is 3% (w/v).

(3) 16s rDNA sequence analysis

The following is the 16S rDNA sequence of strain TGB-11:

AGAGTTTGATCCTGGCTCAGATTGAACGCTGGCGGCAGGCCTAACACATGCAAGTCGAGCGGAAACGGCAGCATTGAAGCTTCGGTGGATTTGCTGGACGTCGAGCGGCGGACGGGTGAGTAACGGCTGGGAACCTGCCCTGACGAGGGGGATAACCGTTGGAAACGACGGCTAATACCGCATAATGTCTTAGTTCATTACGAGCTGGGACCAAAGGTGGCCTCTACATGTAAGCTATCGCGTTGGGATGGGCCCAGTTAGGATTAGCTAGTTGGTAAGGTAATGGCTTACCAAGGCAACGATCCTTAGCTGGTTTGAGAGGATGATCAGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGGGAGACCCTGATGCAGCCATGCCGCGTGTGTGAAGAAGGCCTTCGGGTTGTAAAGCACTTTCAGCAGTGAGGAAGGTGGTGTACTTAATACGTGCATTGCTTGACGTTAGCTGCAGAAGAAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCGAGCGTTAATCGGAATTACTGGGCGTAAAGCGCATGCAGGCGGTTTGTTAAGTCAGATGTGAAAGCCCGGGGCTCAACCTCGGAACCGCATTTGAAACTGGCAGGCTAGAGTCTTGTAGAGGGGGGTAGAATTTCAGGTGTAGCGGTGAAATGCGTAGAGATCTGAAGGAATACCAGTGGCGAAGGCGGCCCCCTGGACAAAGACTGACGCTCAGATGCGAAAGCGTGGGTAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGCTGTCTACTTGGAGGTTGAGGTTTAAGACTTTGGCTTTCGGCGCTAACGCATTAAGTAGACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACC TTACCTACTCTTGACATCCAGAGAACTTTCCAGAGATGGATTGGTGCCTTCGGGAACTCTGAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTTGTGAAATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATCCTTGTTTGCCAGCACGTAATGGTGGGAACTCCAGGGAGACTGCCGGTGATAAACCGGAGGAAGGTGGGGACGACGTCAAGTCATCATGGCCCTTACGAGTAGGGCTACACACGTGCTACAATGGCAGATACAGAGGGCAGCGAAGCTGCGAAGTGGAGCGAATCCCTTAAAGTCTGTCGTAGTCCGGATTGGAGTCTGCAACTCGACTCCATGAAGTCGGAATCGCTAGTAATCGTGGATCAGAATGCCACGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTGGGCTGCACCAGAAGTAGATAGCTTAACCTTCGGGAGGGCGTTTACCACGGTGTGGTTCATGACTGGGGTGAAGTCGTAACAAGGTAACC

The 16S rDNA gene sequence of strain TGB-11 is 1531 bp. The comparison of gene sequences shows that the gene sequence similarity between strain TGB-11 and Salinivibrio strains is relatively high, and the strain with the highest similarity is S.kushneri AL184 (99.68%) .

Based on the above morphological, physiological and biochemical characteristics, and the results of genome sequence determination, it is finally determined that the strain TGB-11 of the present invention is Salinivibrio kushneri.

Strain GTB-11 has been deposited in the General Microbiology Center (CGMCC) of the China Microorganism Culture Collection and Management Committee. Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, zip code 100101. Deposit date November 25, 2020. The deposit number is CGMCC No.21229. Strain name: Salinivibrio; Latin name: Salinivibriokushneri; Ginseng's biological material (strain): TGB-11.

Embodiment 2: Utilize glucose as carbon source to produce polyhydroxyalkanoate

1. Aseptic preparation of S. kushneri TGB-11 seed solution

(1) Strain activation

Take the strain glycerol tube stored in the -80°C refrigerator, streak it to the TYS solid medium plate, and cultivate at 30°C for 16h.

(2) Preparation of seed solution

A single colony was picked from the plate on which the step (1) was completed, inoculated into a TYS liquid medium, and cultured with shaking at 30° C. and 200 rpm for 12 hours to obtain a seed solution.

2. The experiment consists of two treatments: the experimental group and the control group.

Experimental group: The seed solution obtained in step 1 (2) was inoculated into the TYS liquid medium containing 20g/L glucose according to the inoculation amount of 2.5% (that is, 1mL of seed solution, 39mL of culture medium), using a 250mL shaker flask, The total volume of the liquid was 40 mL, and cultured at 30° C. and 200 rpm for 36 h to prepare a fermentation broth.

Control group: In addition, the seed solution obtained in step 1 (2) was inoculated into TYS liquid medium containing no glucose according to 2.5% inoculation volume (1mL of seed solution, 39mL of culture medium), using a 250mL shaker flask, and the total volume of solution The amount was 40 mL, cultured at 30 °C and 200 rpm for 36 h, and a fermentation broth was prepared as a control.

3. Take all the fermentation broth, put it in a 50mL centrifuge tube, centrifuge at 10,000 rpm for 10 minutes, discard the supernatant, resuspend the bacteria with deionized water for washing, and then centrifuge again at 10,000 rpm for 10 minutes to collect the bacteria. The precipitation centrifuge tube was frozen at -20°C for 2 hours, and then placed in a freeze-drying machine for 10 hours to obtain a freeze-dried product. Accurately weigh the centrifuge tube before adding the fermentation broth and after lyophilization, and calculate the dry cell weight.

4. Transfer the lyophilized cells to an esterification tube, weigh the dry weight of the transferred cells (about 30-40 mg), add 2 mL of esterification solution and 2 mL of chloroform, seal with a lid, and react in an oven at 100 °C 4h; after cooling to room temperature, add 1 mL of deionized water, fully shake, stand for stratification; after the chloroform phase and the water phase are completely separated, take the chloroform phase and conduct gas chromatography analysis;

Using Agilent's micro-injector, the injection volume was 1 μL, and the internal standard method was used to quantitatively analyze the polymer, which was quantified according to the peak area.

The poly-3-hydroxybutyrate yield algorithm is: PHB yield = (PHB peak area in the sample/internal standard peak area in the sample) × [(internal standard peak area in the standard product/PHB peak area in the standard product) × (standard product peak area product mass×0.866)]/sample esterification mass×cell dry weight

Through calculation, in the experimental group, when glucose was the carbon source, the dry cell weight was 6.35 g/L, and the polyhydroxyalkanoate accumulated in the cells was poly-3-hydroxybutyrate, and the yield was 5.00 g/L, accounting for 5.00 g/L of cell dry weight. The heavy mass fraction is 78.71%.

In the control group, when no glucose was added, the dry weight of the cells was 1.44 g/L, and no polyhydroxyalkanoate was produced.

In addition, the bacteria in Example 2 were observed after culturing with a transmission electron microscope. The results are shown in Figure 1 and Figure 2, respectively, where the scales are all 200 nm. In Figure 1, when glucose was used as the carbon source, the bacteria accumulated a large number of white polyhydroxyalkanoate particles in the cells. In Figure 2, when no glucose was added, a large number of white polyhydroxyalkanoate particles were not observed in the cells.

Example 3: Production of polyhydroxyalkanoate with maltose as substrate

The specific operation is according to Example 2 of the present invention, except that the medium in the experimental group is a liquid TYS medium containing 20 g/L maltose.

Through calculation, when maltose is the carbon source, the dry cell weight is 5.82g/L, and the polyhydroxyalkanoate accumulated in the cell is poly-3-hydroxybutyrate, and the yield is 3.73g/L, accounting for the mass of the cell dry weight. The score is 64.16%.

Example 4: Production of polyhydroxyalkanoate with soluble starch as carbon source

The specific operation was in accordance with Example 2 of the present invention, except that the medium in the experimental group was a liquid TYS medium containing 20 g/L soluble starch.

By calculation, when soluble starch is the carbon source, the dry weight of cells is 6.78g/L, and the polyhydroxyalkanoate accumulated in cells is poly-3-hydroxybutyrate, and the yield is 3.28g/L, accounting for 3.28g/L of dry cell weight. The quality score is 48.36%.

Example 5: Production of polyhydroxyalkanoate with sucrose as carbon source

The specific operation was in accordance with Example 2 of the present invention, except that the medium in the experimental group was a liquid TYS medium containing 20 g/L sucrose.

By calculation, when sucrose is the carbon source, the dry cell weight is 5.62 g/L, and the polyhydroxyalkanoate accumulated in the cells is poly-3-hydroxybutyrate, and the yield is 3.23 g/L, accounting for the mass of the dry cell weight. The score is 57.46%.

Example 6: Production of polyhydroxyalkanoate with acetic acid as carbon source

The specific operation was in accordance with Example 2 of the present invention, except that the medium in the experimental group was a liquid TYS medium containing 20 g/L acetic acid.

By calculation, the dry weight of cells when acetic acid is the carbon source is 3.90g/L, the polyhydroxyalkanoate accumulated in the cells is poly-3-hydroxybutyrate, and the yield is 0.77g/L, accounting for the mass of dry cells. The score is 19.72%.

Example 7: Production of polyhydroxyalkanoate with butyric acid as carbon source

The specific operation is according to Example 2 of the present invention, except that the medium in the experimental group is a liquid TYS medium containing 10 g/L butyric acid.

By calculation, when butyric acid is the carbon source, the cell dry weight is 2.90g/L, and the polyhydroxyalkanoate accumulated in the cells is poly-3-hydroxybutyrate, and the yield is 0.88g/L, accounting for 3% of the cell dry weight. The mass fraction is 30.34%.

Example 8: Production of polyhydroxyalkanoate with glucose and propionic acid as mixed carbon sources

The specific operation is according to Example 2 of the present invention, the difference is that the culture medium in the experimental group is a medium containing 20g/L glucose and different concentrations of propionic acid (0.5g/L, 1g/L, 1.5g/L or 2g/L). Liquid TYS medium; accumulation of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) was detected and calculated in step 4. The experimental results are shown in Table 1.

Table 1 Production of polyhydroxyalkanoate with glucose and propionic acid as mixed carbon sources

When glucose and propionic acid were used as mixed carbon sources, poly(3-hydroxybutyrate-co-3-hydroxyvalerate) could be obtained by fermentation and culture, the dry weight of cells decreased with the increase of propionic acid concentration, and the production of PHBV And the proportion of dry weight of cells decreased with the increase of propionic acid concentration, and the monomer content of 3-hydroxyvaleric acid increased with the increase of propionic acid concentration, and the highest was more than 42mol%. When 0.5g/L propionic acid was added, the yield of PHBV was the highest, the yield was 4.38g/L, and the content of 3-hydroxyvaleric acid monomer was 8.57mol%.

Example 9: Production of polyhydroxyalkanoates with different disaccharides and propionic acid as mixed carbon sources

The specific operation is according to Example 2 of the present invention, the difference is that the medium in the experimental group is a liquid TYS medium containing 1.5g/L propionic acid and 20g/L different disaccharides (sucrose, maltose or soluble starch); Step 4 The accumulation of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) was detected in and calculated. The experimental results are shown in Table 2.

Table 2 Production of polyhydroxyalkanoates with different disaccharides and propionic acid as mixed carbon sources

Using sucrose, maltose and soluble starch added with 1.5 g/L propionic acid as mixed carbon sources respectively, poly(3-hydroxybutyrate-co-3-hydroxyvalerate) can be obtained by fermentation culture. The dry weight of cells is all above 7g/L, and the content of 3-hydroxyvaleric acid monomer is 17.40mol%-30.00mol%. Among the three different disaccharides, sucrose had the best effect, and the yield of PHBV was 4.62 g/L. Considering that sucrose is a raw material with a wide range of sources, the strain provided by the present invention has a good application prospect in producing PHBV by utilizing sucrose and propionic acid.

Example 10: Production of polyhydroxyalkanoate with glucose and seawater as raw materials

The specific operation is according to Example 2 of the present invention, the difference is that the culture medium in the experimental group is a natural seawater culture medium (ammonium sulfate 10g/L, dipotassium hydrogen phosphate 10g/L, natural seawater culture medium containing 20g/L glucose) The solvent is seawater, and the seawater is taken from the coastal waters of Binhai New Area, Tianjin).

By calculation, when glucose and seawater are used as raw materials, the dry weight of cells is 3.03g/L, the polyhydroxyalkanoate accumulated in cells is poly-3-hydroxybutyrate, and the yield is 2.64g/L, accounting for 2.64g/L of dry cell weight. The quality score is 87.13%.

SEQUENCE LISTING

<110> Beijing University of Chemical Technology

<120> A salt vibrio and its application

<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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