CN111206058B - A kind of method utilizing acetic acid or butyric acid to produce polyhydroxyalkanoic acid ester - Google Patents

Abstract

The invention discloses a method for producing polyhydroxyalkanoate by using acetic acid or butyric acid. According to the invention, the hyphomonas with polyhydroxyalkanoate production capacity can utilize acetic acid or butyric acid as a carbon source, a large amount of polyhydroxyalkanoate is accumulated under an open condition without sterilization, the proportion of polyester in the dry weight of bacteria can reach 69% at most, and the yield with butyric acid as the carbon source can reach 40% of the theoretical yield at most. The method produces the polyhydroxyalkanoate by two unusual carbon sources of butyric acid or acetic acid, does not need sterilization in the fermentation process, can reduce the fermentation cost of the polyhydroxyalkanoate, and has great economic advantages and good industrial application prospects.

Description

A kind of method utilizing acetic acid or butyric acid to produce polyhydroxyalkanoic acid ester

technical field

The invention belongs to the field of biochemistry, relates to a method for producing polyhydroxy fatty acid ester, and more particularly relates to a method for producing polyhydroxy fatty acid ester by utilizing acetic acid or butyric acid.

Background technique

Polyhydroxyalkanoates (PHA) is a kind of macromolecular polymer synthesized in cells by microorganisms under the condition of unbalanced growth and metabolism. It is mainly used as carbon source and energy storage material. Microbial re-decomposition and utilization. PHA has similar material properties as petroleum-derived plastic materials, and at the same time has many excellent properties that petroleum-derived plastic materials lack, such as biorenewability, biodegradability, biocompatibility and so on. Over the years, the synthesis and utilization of PHA have attracted extensive attention from the scientific and industrial circles. A lot of research work has been carried out in reducing the production cost of PHA, synthesizing PHA materials with new monomer compositions and structures, and developing a variety of high value-added applications of PHA, and a series of important research results have been achieved.

At present, the production cost of PHA is still high, which limits its large-scale industrial production and application. The use of relatively cheap substrates to replace carbon sources such as glucose widely used in industrial fermentation will help reduce the dependence of fermentation raw materials on grain crops, and solve the problems of competing with people for grain and grain, which is of great significance. Volatile fatty acids, including acetic acid and butyric acid, can be prepared by the transformation of various biomass wastes by microorganisms. In recent years, they have been regarded as a kind of fermentation raw material with good application prospects.

SUMMARY OF THE INVENTION

The object of the present invention is to provide a method for producing polyhydroxyalkanoate by utilizing acetic acid or butyric acid.

The present invention claims the application of Poseidononas in the preparation of polyhydroxyalkanoates.

Specifically, the substrates used are non-sugar raw materials or volatile fatty acids; more specifically, butyric acid or acetic acid.

The present invention also claims a method for preparing polyhydroxyalkanoate, the method comprising:

Using the aforementioned substrate, fermented and cultured Poseidonella to obtain the polyhydroxyalkanoate.

In the above method, the concentration of the substrate in the system at the start of fermentation is 10-20 g/L.

Specifically, the concentration of the butyric acid in the system at the start of fermentation is 10 g/L.

The concentration of the acetic acid in the system at the start of fermentation was 20 g/L.

At the initial moment of fermentation, the OD ₆₀₀ value of the system containing the Neptune bacterium is 0.05-0.25; specifically, 0.1;

The initial time of the fermentation is the initial time after the seed liquid is inoculated into the fermentation medium;

In the fermentation and culture step, the fermentation temperature is 37°C;

Dissolved oxygen is 10-50%; specifically 30%;

The stirring speed is 200-800rpm;

The fermentation time is 36-60h; more specifically 48h;

pH value is 7-9; specifically 8;

The ventilation volume of air is 1-5L/min; specifically 3L/min.

The fermentation is liquid fermentation;

The fermentation culture is an open culture; the open culture is specifically an open bottle or in a non-sterile environment (such as a bottle) covered with a sealing film, or in a culture medium and a fermenter without sterilization and no filtration is required for passage. Cultured in sterile air fermenters;

In the fermentation, the fermentation system consists of a seed liquor, a culture medium and the substrate;

The active ingredient provided by the seed liquid is the Poseidononas;

The cultivation of the seed liquid is aseptic operation cultivation;

Described culture medium is liquid culture medium; In particular, it is any culture medium that can be used for described Poseidonium;

More specifically, it can be a liquid TYS medium composed of the following proportions: 5 g of peptone, 1 g of yeast powder, and the volume is adjusted to 1 L with artificial seawater;

Wherein, the artificial seawater is composed of the following components in the following proportions: 27.5g of sodium chloride, 0.7g of potassium chloride, 5.4g of magnesium chloride hexahydrate, 6.8g of magnesium sulfate heptahydrate, 1.05g of calcium chloride, 0.2g of sodium bicarbonate g, make up to 1L with deionized water.

Described seed liquid can be obtained according to the following method:

a, bacterial activation

Take the strain glycerol tube stored in the -80°C refrigerator, streak it to the TYS medium plate, and cultivate it at 37°C for 12-24h;

b, first-class seed liquid

Pick a single colony from the plate that has completed the step a, inoculate it in a liquid TYS medium, and shake it at 37°C at 200rpm for 10-12h to obtain a first-class seed solution;

c, secondary seed solution

Take the first-class seed solution, inoculate it in liquid TYS medium according to the inoculum amount of 1%, add 10g/L acetic acid or 10g/L butyric acid as a carbon source, 37 ℃, 200rpm shaking culture for 10-12h, get the second-class seed fluid.

In the present invention, the polyhydroxyalkanoic acid ester may specifically be poly-3-hydroxybutyrate, copolyester of 3-hydroxybutyric acid and 3-hydroxyvaleric acid, or copolyester of 3-hydroxybutyric acid and 4-hydroxybutyric acid Polyester; the copolyester of 3-hydroxybutyric acid and 3-hydroxyvaleric acid is an ester obtained by copolymerizing 3-hydroxybutyric acid and 3-hydroxyvaleric acid; the 3-hydroxybutyric acid and 4-hydroxybutyric acid Acid copolyesters are esters obtained by copolymerizing 3-hydroxybutyric acid and 4-hydroxybutyric acid.

The Neptunomonas concharum of the present invention is isolated from the marine environment and can grow well under the condition of NaCl concentration of 25-50 g/L. Different from the lower optimal growth temperature of common marine microorganisms, N. concharum JCM17730 can grow well under the conditions of 30-37 ℃ in common industrial fermentation. Different from common fermentation industrial strains such as Escherichia coli, Lactobacillus, Corynebacterium glutamicum and Saccharomyces cerevisiae, N.concharum JCM17730 has weak utilization of carbohydrate carbon sources such as glucose, fructose and sucrose, but can utilize acetic acid, propionic acid and Volatile fatty acids such as butyric acid are used as carbon sources for rapid growth.

In the present invention, Poseidononas with the ability to produce polyhydroxyalkanoates can use acetic acid or butyric acid as a carbon source to accumulate a large amount of polyhydroxyalkanoates under open conditions without sterilization, and polyester occupies bacteria The dry weight ratio can reach up to 69%, and the yield with butyric acid as the carbon source can reach up to 40% of the theoretical yield. The method uses two uncommon carbon sources, acetic acid or butyric acid, to produce polyhydroxyalkanoates, without sterilization in the fermentation process, can reduce the fermentation cost of polyhydroxyalkanoates, and has great economic advantages and good industrial applications prospect.

Description of drawings

Fig. 1 is a fermentor culture for the production of polyhydroxyalkanoate by using butyric acid by Poseidon.

Detailed ways

The present invention is further described below in conjunction with specific embodiments, but the present invention is not limited to the following embodiments. The methods are conventional methods unless otherwise specified. The raw materials can be obtained from open commercial sources unless otherwise specified. The quantitative experiments in the following examples were all set to repeat the experiments three times, and the results were averaged. The standard product of polyhydroxyalkanoate was purchased from Sigma-Aldrich, the product number is 403121, the product name is poly(3-hydroxybutyric acid-co-3-hydroxyvaleric acid), and the content of poly-3-hydroxybutyric acid is 88 mol%. The Neptunomonas concharum used in the following examples can be purchased from the Japan Collection of Microorganisms, and the representative strain is preserved in the Japan Collection of Microorganisms, and the preservation number is JCM17730.

The method for preparing seed liquid in the embodiment is as follows:

a, bacterial activation

Take the strain glycerol tube stored in the -80°C refrigerator, streak it to the TYS medium plate, and cultivate it at 37°C for 12-24h;

b, first-class seed liquid

Pick a single colony from the plate that has completed the step a, inoculate it in a liquid TYS medium, and shake it at 37°C at 200rpm for 10-12h to obtain a first-class seed solution;

c, secondary seed solution

Take the first-class seed solution, inoculate it in liquid TYS medium according to the inoculum amount of 1%, add 10g/L acetic acid or 10g/L butyric acid as a carbon source, 37 ℃, 200rpm shaking culture for 10-12h, get the second-class seed fluid.

The method of freeze-drying in the embodiment:

After fermentation with acetic acid or butyric acid as the carbon source, take 30ml of fermentation broth, centrifuge at 8000rpm for 10min, discard the supernatant, resuspend the cells with deionized water, wash, and centrifuge again at 8000rpm for 10min to collect the cells and freeze-dry them (Place the centrifuge tube containing the washed cell precipitation at -20°C for 1 hour, and then put it into a freeze-drying device for 8-12 hours) to obtain a freeze-dried product;

In the examples, the dry cell weight is measured as the dry cell weight per liter of fermentation broth. The unit of cell dry weight is g/L. Cell dry weight (CDW for short) = (the weight of the centrifuge tube after freeze-drying - the weight of the original empty centrifuge tube) ÷ 0.03; the weight of the centrifuge tube after freeze-drying and the weight of the original empty centrifuge tube, The unit is g; 0.03 means that the volume of fermentation broth is 0.03L.

The detection method for the content of the polyhydroxyalkanoate in the bacterial cells in the examples: the freeze-dried product is subjected to esterification, and then the monomer content is determined by gas chromatography.

Esterification reaction: take 50mg of freeze-dried product in an esterification tube, add 2mL of chloroform and 2mL of esterification solution (the esterification solution is obtained by adding 15ml of concentrated sulfuric acid and 1g of benzoic acid to 500ml of methanol), mix well, cover and seal , esterified at 100°C for 4 hours; after cooling to room temperature, add 1 mL of deionized water, fully shake and mix with a vortex shaker, and let stand for stratification; after the chloroform phase and water are completely separated, take 1 μL of the chloroform phase for gas chromatography analyze.

20 mg of poly(3-hydroxybutyric acid-co-3-hydroxyvaleric acid) was taken and subjected to an esterification reaction in the same manner as a standard sample.

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; increase the temperature to 140°C at a rate of 30°C/min and stay for 0min;

The temperature was raised to 220°C at a rate of 40°C/min and held for 1 min. 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.

During gas chromatography detection, the freeze-dried cell sample was compared with the poly(3-hydroxybutyric acid-co-3-hydroxyvaleric acid) standard, and the freeze-dried cell sample contained the peak at the position of 3-hydroxybutyric acid in the standard. At the same time, take the freeze-dried cell sample and add it to chloroform solution, keep it in a 100 ℃ oven for 4 hours, after cooling, take the chloroform phase, add 6 times the volume of ethanol, a white precipitate is precipitated; the precipitate is taken out, and the above steps are used to carry out esterification reaction and gas chromatography Detection, the peak position is the same as that of 3-hydroxybutyric acid in the standard. This indicates that the polyhydroxyalkanoate accumulated in the bacterial cells is poly-3-hydroxybutyrate.

Polymer content is defined as the ratio of polymer to dry cell weight, polymer yield = polymer content x dry cell weight.

In the following examples, butyric acid was quantitatively detected by high performance liquid chromatography according to the following method.

The specific conditions are as follows:

Instrument: Shimadzu Essentia LC series HPLC instrument, equipped with DGU-20A degasser, LC-16 liquid pump, SIL-16 type autosampler, RID-20A detector.

HPX-87H(7.8×300mm)；流速0.50mL/min；柱温55℃；流动相为7mM硫酸水溶液。Chromatographic conditions: Bio-Rad

HPX-87H (7.8×300mm); flow rate 0.50mL/min; column temperature 55°C; mobile phase is 7mM aqueous sulfuric acid.

Detection method:

Take 0, 1, 2, 3, 4, and 5 g/L of butyric acid standard solution (Sigma-Aldrich, product number B103500), filter with 0.22 μm microporous membrane, inject 10 μL of sample, carry out HPLC detection, use different concentrations The chromatographic peak area of the butyric acid standard solution is the ordinate, and the different concentrations are the abscissa, and the standard curve is drawn.

Take 2 mL of the fermentation broth, centrifuge at 12000 rpm for 10 min, transfer the fermentation supernatant to a new centrifuge tube, filter with a 0.22 μm microporous membrane, inject 10 μL of the sample, and perform HPLC detection.

Substitute the butyric acid chromatographic peak area of the fermentation supernatant of the sample to be tested into the above standard curve, and calculate the butyric acid content of the fermentation supernatant of the sample to be tested.

In the following examples, at the initial moment of fermentation, the OD ₆₀₀ values of the systems containing Neptuneia were all 0.1.

Embodiment 1, utilizes acetic acid to produce polyhydroxyalkanoate (shaker flask culture) with Poseidon

1. Using acetic acid to produce polyhydroxy fatty acid ester by Poseidon

1. Preparation of Poseidononas seed solution by aseptic operation

(1) Strain activation

Take the strain glycerol tube stored in the -80°C refrigerator, streak it to the TYS medium plate, and cultivate at 37°C for 12-24 hours.

(2) First-class seeds

Pick a single colony from the plate that has completed step (1), inoculate it in a liquid TYS medium, and cultivate with shaking at 200 rpm at 37° C. for 10-12 hours.

(3) Secondary seeds

The first-grade seed liquid obtained in step (2) was inoculated into liquid TYS medium according to the inoculum amount of 1%, 10 g/L acetic acid was added as a carbon source, and 37° C., 200 rpm was shaken for 10-12 h.

2. Prepare liquid TYS medium, add acetic acid to the final concentration of 20g/L, and adjust the pH to 7.

In addition, follow the same steps as above, and set the following comparison:

The acetic acid was replaced by propionic acid, and the concentration was replaced by 10g/L;

3. The seed solution obtained in step 1 (3) was inoculated into the liquid TYS medium containing 20g/L acetic acid according to the inoculum amount of 8% (ie, secondary seed solution 4ml, culture medium 46ml), and a 250ml shake flask was used. , the total volume of liquid is 50ml, and cultured at 37°C and 200rpm for 40-48h to prepare a fermentation broth.

In addition, follow the same steps as above, and set the following comparison:

The acetic acid was replaced by propionic acid, and the concentration was replaced by 10g/L;

4. Take 30ml of fermentation broth, put it in a centrifuge tube with a volume of 50ml, and centrifuge at 8000rpm for 10min. After discarding the supernatant, resuspend and wash the cells with deionized water. Centrifuge again at 8000rpm for 10min. After discarding the supernatant, put the cells in It was placed at -20°C for 1 hour, and then vacuum freeze-dried for 8-12 hours to obtain freeze-dried cells. Accurately weigh the centrifuge tube before adding the fermentation broth and after lyophilization, and calculate the dry cell weight.

5. Transfer the freeze-dried thalline to an esterification tube of known quality, weigh the dry weight of the transferred thalli, add 2 mL of esterification solution (3% by volume, and dissolve concentrated sulfuric acid in methanol). , obtain a sulfuric acid-methanol solution, and then add benzoic acid as an internal standard to the sulfuric acid-methanol solution according to the final concentration of 1 g/L to obtain an esterification solution), 2 mL of chloroform, cover and seal, and react in a 100 ℃ oven for 4 h; After room temperature, 1 mL of deionized water was added, fully shaken, and allowed to stand for stratification; after the chloroform phase was completely separated from the water phase, the chloroform phase was taken and analyzed by gas chromatography;

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

Polymer content is defined as the ratio of polymer to dry cell weight, polymer yield = polymer content x dry cell weight.

By calculation, when acetic acid was obtained as the substrate, the dry weight of the cells was 3.95 g/L, the polyhydroxyalkanoate accumulated in the cells was poly-3-hydroxybutyrate, and the yield was 1.62 g/L.

As a control, when propionic acid was used as the substrate, the dry weight of the cells was 1.96 g/L, and no polyester was produced.

2. Open culture without inoculation of TYS acetate medium

1. Prepare liquid TYS medium, add acetic acid to the final concentration of 20g/L, and adjust the pH to 7. Do not inoculate any microorganisms, use a 250ml shake flask with a total liquid volume of 50ml, culture at 37°C and 200rpm for 40-48h to prepare a fermentation broth.

2. Since there is no inoculation, but there are miscellaneous bacteria growing under open conditions, according to the method of the above one, the dry weight of cells and the yield of polyhydroxyalkanoate in the culture of miscellaneous bacteria are detected.

It was found that the dry cell weight was 0.17 g/L, and no accumulation of polyhydroxyalkanoates was detected. This shows that with 20g/L of acetic acid as the carbon source, the general microorganisms rarely grow, and the biomass of miscellaneous bacteria is very low.

3. Shake flask culture experiment of Poseidonella under acetic acid-free conditions

1. According to the above method, replace the fermentation medium with TYS medium without acetic acid (the difference from TYS acetic acid medium is only that the components do not contain acetic acid), and other steps remain unchanged.

2. The dry weight of cells and the accumulation of polyhydroxyalkanoates were detected. The results showed that the dry weight of cells was 1.12 g/L after Poseidononas was cultured in a shake flask without acetic acid, and no polyhydroxyalkanoates were detected. accumulation.

It can be seen that only the peptone and yeast extract in the TYS medium is not enough to ensure the production of polyhydroxyalkanoates by Poseidomonas, and at the same time, there should be miscellaneous bacteria growth in the fermentation without sterilization.

Embodiment 2, utilizes butyric acid to produce polyhydroxyalkanoic acid ester (shaking flask culture) with Poseidon

1. Using butyric acid to produce polyhydroxy fatty acid ester by Poseidon

1. Preparation of Poseidononas seed solution by aseptic operation

(1) Strain activation

Take the strain glycerol tube stored in the -80°C refrigerator, streak it to the TYS medium plate, and cultivate at 37°C for 12-24 hours.

(2) First-class seeds

Pick a single colony from the plate that has completed step (1), inoculate it in a liquid TYS medium, and cultivate with shaking at 200 rpm at 37° C. for 10-12 hours.

(3) Secondary seeds

The first-grade seed solution obtained in step (2) was inoculated into liquid TYS medium according to the inoculum amount of 1%, 10 g/L butyric acid was added as a carbon source, and 37° C., 200 rpm was shaken for 10-12 h.

2. Prepare liquid TYS medium, add butyric acid to a final concentration of 10 g/L, and adjust pH to 7.

3. Inoculate the seed solution obtained in step 1(3) into a liquid TYS medium containing butyric acid according to the inoculum volume of 8%, use a 250ml shake flask, the total volume of liquid is 50ml, and cultivate at 37°C and 200rpm for 40-48h , to make fermentation broth.

4. Take 30ml of fermentation broth, put it in a centrifuge tube with a volume of 50ml, and centrifuge at 8000rpm for 10min. After discarding the supernatant, resuspend and wash the cells with deionized water. Centrifuge again at 8000rpm for 10min. After discarding the supernatant, put the cells in It was placed at -20°C for 1 hour, and then vacuum freeze-dried for 8-12 hours to obtain freeze-dried cells. Accurately weigh the centrifuge tube before adding the fermentation broth and after lyophilization, and calculate the dry cell weight.

5. Transfer the freeze-dried thalline to an esterification tube of known quality, weigh the dry weight of the transferred thalli, add 2 mL of esterification solution (3% by volume, and dissolve concentrated sulfuric acid in methanol). , obtain a sulfuric acid-methanol solution, and then add benzoic acid as an internal standard to the sulfuric acid-methanol solution according to the final concentration of 1 g/L to obtain an esterification solution), 2 mL of chloroform, cover and seal, and react in a 100 ℃ oven for 4 h; After room temperature, 1 mL of deionized water was added, fully shaken, and allowed to stand for stratification; after the chloroform phase was completely separated from the water phase, the chloroform phase was taken and analyzed by gas chromatography;

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

Polymer content is defined as the ratio of polymer to dry cell weight, polymer yield = polymer content x dry cell weight.

By calculation, when butyric acid was obtained as the substrate, the dry weight of the cells was 5.01g/L, the polyhydroxyalkanoate accumulated in the cells was poly-3-hydroxybutyrate, and the yield was 3.49g/L, accounting for the dry weight of the cells. 69%. It can be seen that butyric acid can obtain higher dry cell weight and polyhydroxyalkanoate yield than acetic acid. After calculation, the theoretical maximum yield of synthesizing poly-3-hydroxybutyrate with butyric acid as carbon source is 0.98g poly-3-hydroxybutyrate/g butyric acid, and the actual yield obtained in this example has reached the theoretical yield. 40% of the rate.

2. Open culture without inoculation in TYS butyric acid medium

1. Prepare liquid TYS medium, add butyric acid to a final concentration of 20g/L, and adjust pH to 7. Do not inoculate any microorganisms, use a 250ml shake flask with a total liquid volume of 50ml, culture at 37°C and 200rpm for 40-48h to prepare a fermentation broth.

2. Since there is no inoculation, but there are miscellaneous bacteria growing under open conditions, according to the method of the above one, the dry weight of cells and the yield of polyhydroxyalkanoate in the culture of miscellaneous bacteria are detected.

It was found that the dry cell weight was 0.06 g/L, and no accumulation of polyhydroxyalkanoates was detected. This shows that with 10g/L butyric acid as the carbon source, the general microorganisms rarely grow, and the biomass of miscellaneous bacteria is very low.

Embodiment 3, utilizes butyric acid to produce polyhydroxyalkanoate (fermentor culture) with Poseidon

1. Using butyric acid to produce polyhydroxy fatty acid ester by Poseidon

1. Preparation of Poseidononas seed solution by aseptic operation

(1) Strain activation

Take the strain glycerol tube stored in the -80°C refrigerator, streak it to the TYS medium plate, and cultivate at 37°C for 12-24h.

(2) First-class seeds

Pick a single colony from the plate that has completed step (1), inoculate it in a liquid TYS medium, and cultivate with shaking at 200 rpm at 37° C. for 10-12 hours.

(3) Secondary seeds

The first-grade seed liquid obtained in step (2) was inoculated into liquid TYS medium according to the inoculum amount of 1%, 10 g/L butyric acid was added as a carbon source, and it was shaken and cultured at 37° C. and 200 rpm for 10-12 h. A total of 300ml of secondary seed liquid required for fermenter culture.

2. Make 2700ml TYS medium, add butyric acid to the final concentration of 10g/L. The medium was added to a fermenter with a volume of 5 L (Bai Lun Bio, model BLBIO-5GJ-2).

3. Add 300ml of secondary seed liquid into the fermenter, make the fermentation system 3L, the fermentation temperature is 37°C, keep the dissolved oxygen at 30% through the associated stirring rate, and the stirring rate is 400-800rpm, using 6M sodium hydroxide and 3M The pH value was adjusted with hydrochloric acid, and the fermentation was carried out continuously under the condition of pH value of 8, and the ventilation rate of air was 3L/min. Every 4h, 50ml was sampled and kept for testing the dry weight of cells and the production of polyhydroxyalkanoate. At the same time, the amount of butyric acid was detected in real time, and butyric acid was supplemented, and the fermentation was carried out for 52h.

5. Take 30ml of fermentation broth, put it in a centrifuge tube with a volume of 50ml, and centrifuge at 8000rpm for 10min. After discarding the supernatant, resuspend and wash the cells with deionized water. Centrifuge again at 8000rpm for 10min. After discarding the supernatant, put the cells in It was placed at -20°C for 1 hour, and then vacuum freeze-dried for 8-12 hours to obtain freeze-dried cells. Accurately weigh the centrifuge tube before adding the fermentation broth and after lyophilization, and calculate the dry cell weight.

6. Transfer the freeze-dried thalli to an esterification tube of known quality, weigh the dry weight of the transferred thalli, add 2 mL of esterification solution (3% by volume, and dissolve concentrated sulfuric acid in methanol). , obtain a sulfuric acid-methanol solution, and then add benzoic acid as an internal standard to the sulfuric acid-methanol solution according to the final concentration of 1 g/L to obtain an esterification solution), 2 mL of chloroform, cover and seal, and react in a 100 ℃ oven for 4 h; After room temperature, 1 mL of deionized water was added, fully shaken, and allowed to stand for stratification; after the chloroform phase was completely separated from the water phase, the chloroform phase was taken and analyzed by gas chromatography;

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

8. The polymer content is defined as the ratio of polymer to dry cell weight, polymer yield = polymer content × dry cell weight.

The results showed (Fig. 1) that the dry weight of cells and the yield of polyhydroxyalkanoate reached the highest at 48 h of fermentation, the dry cell weight was 35.07 g/L, and the polyester accumulated in the cells was poly-3-hydroxybutyrate. was 15.51 g/L, the total amount of butyric acid consumed by fermentation was 54.17 g/L, and the fermentation yield was 0.29 g poly-3-hydroxybutyrate/g butyric acid. After calculation, the theoretical maximum yield of synthesizing poly-3-hydroxybutyrate with butyric acid as carbon source is 0.98g poly-3-hydroxybutyrate/g butyric acid, and the actual yield obtained in this example has reached the theoretical yield. 30% of the rate.

Claims (11)

1. The application of the hyphosporon in preparing polyhydroxyalkanoate;

the number of the hyphosporon is JCM 17730;

the substrate is acetic acid or butyric acid.

2. Use according to claim 1, characterized in that: the polyhydroxyalkanoate is poly-3-hydroxybutyrate.

3. A method of making a polyhydroxyalkanoate comprising:

fermenting and culturing the hyphomonas by taking acetic acid or butyric acid as a substrate to obtain the polyhydroxyalkanoate;

the number of the sea-deinsectization monads is JCM 17730.

4. The method of claim 3, wherein: the concentration of the substrate in the system at the beginning of the fermentation is 10-20 g/L.

5. The method of claim 3, wherein: the concentration of butyric acid in the system at the start of fermentation was 10 g/L.

6. The method of claim 3, wherein: the concentration of acetic acid in the system at the start of fermentation was 20 g/L.

7. The method according to any one of claims 3-6, wherein: at the initial stage of fermentation, the OD of the system containing said Hippocampus fungi₆₀₀A value of 0.05-0.25;

in the fermentation culture step, the fermentation temperature is 37 ℃;

dissolved oxygen is 10-50%;

the fermentation time is 36-60 h;

the pH value is 7-9;

the ventilation capacity of the air is 1-5L/min.

8. The method of claim 7, wherein: at the initial stage of fermentation, the OD of the system containing said Neurospora₆₀₀A value of 0.1;

in the fermentation culture step, the dissolved oxygen is 30%;

the fermentation time is 48 h;

the pH value is 8;

the ventilation volume of the air is 3L/min.

9. The method according to any one of claims 3-6, wherein: the fermentation culture is open culture;

in the fermentation, a fermentation system consists of a seed solution, a culture medium and the substrate;

the active ingredient provided by the seed liquid is the hyphosporon.

10. The method according to any one of claims 3-6, wherein: the culture of the seed liquid is aseptic culture.

11. The method according to any one of claims 3-6, wherein: the polyhydroxyalkanoate is poly-3-hydroxybutyrate.

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