CN111206058B - Method for producing polyhydroxyalkanoate by using acetic acid or butyric acid - Google Patents
Method for producing polyhydroxyalkanoate by using acetic acid or butyric acid Download PDFInfo
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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
Technical Field
The invention belongs to the field of biochemistry, relates to a method for producing polyhydroxyalkanoate, and more particularly relates to a method for producing polyhydroxyalkanoate by using acetic acid or butyric acid.
Background
Polyhydroxyalkanoates (PHA) is a high molecular polymer synthesized by microorganisms in cells under the condition of unbalanced growth and metabolism, is mainly used as a storage substance of a carbon source and an energy source, and can be decomposed and utilized by the microorganisms again when the nutrition is deficient. PHAs have material properties similar to petroleum-derived plastic materials, while possessing many of the superior properties lacking in petroleum-derived plastic materials, such as biorenewability, biodegradability, biocompatibility, and the like. The synthesis and utilization of PHAs has been a widespread concern in the scientific and industrial sectors for many years. Many research works are carried out on the aspects of reducing the production cost of PHA, synthesizing PHA materials with new monomer compositions and structures, developing high-value-added applications of various PHAs and the like, and a series of important research results are obtained.
At present, the production cost of PHA is still high, which limits the large-scale industrial production and application. The cheap substrate is used for replacing carbon sources such as glucose and the like widely applied in industrial fermentation, the dependence of fermentation raw materials on grain crops is reduced, the problem of competing with people for grain and competing with grain for land is solved, and the method has important significance. Volatile fatty acids including acetic acid and butyric acid, which can be prepared by converting various biomass wastes by microorganisms, have been recently considered as a fermentation raw material with good application prospects.
Disclosure of Invention
The object of the present invention is to provide a method for producing polyhydroxyalkanoate using acetic acid or butyric acid.
The invention claims the application of the hyphosporon in preparing polyhydroxyalkanoate.
Specifically, the substrate is non-sugar raw material or volatile fatty acid; more specifically butyric acid or acetic acid.
The present invention also claims a method for preparing polyhydroxyalkanoate, the method comprising:
fermenting and culturing the hyphosporon by using the substrate to obtain the polyhydroxyalkanoate.
In the above method, the concentration of the substrate in the system at the start of fermentation is 10 to 20 g/L.
Specifically, the concentration of butyric acid in the system at the beginning of fermentation is 10 g/L.
The concentration of acetic acid in the system at the start of fermentation was 20 g/L.
At the initial stage of fermentation, the OD of the system containing said Neurospora600A value of 0.05-0.25; specifically 0.1;
the initial fermentation time is the initial time after the seed solution is inoculated to a fermentation culture medium;
in the fermentation culture step, the fermentation temperature is 37 ℃;
dissolved oxygen is 10-50%; in particular 30 percent;
the stirring speed is 200-800 rpm;
the fermentation time is 36-60 h; more specifically 48 h;
the pH value is 7-9; in particular 8;
the ventilation capacity of the air is 1-5L/min; in particular to 3L/min.
The fermentation is liquid fermentation;
the fermentation culture is open culture; the open culture is specifically an open rock flask or a culture in an environment (such as a rock flask) with a sealing film covering the non-sterile environment or a fermentation tank with a culture medium and a fermentation tank body which are not sterile and into which air without filter sterilization is introduced;
in the fermentation, a fermentation system consists of a seed solution, a culture medium and the substrate;
the effective component provided by the seed liquid is the hyphomomonas;
the culture of the seed liquid is aseptic culture;
the culture medium is a liquid culture medium; in particular to any culture medium which can be used for culturing the hyphosporon;
more specifically, the culture medium can be a liquid TYS culture medium composed of the following components in proportion: 5g of peptone and 1g of yeast powder, and metering the volume to 1L by using artificial seawater;
the artificial seawater comprises the following components in parts by weight: 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 and 0.2g of sodium bicarbonate, and the volume is fixed to 1L by using deionized water to obtain the calcium carbonate.
The seed liquid can be prepared by the following method:
a. strain activation
Taking a strain glycerin tube stored in a refrigerator at the temperature of-80 ℃, streaking and inoculating the strain glycerin tube to a TYS culture medium plate, and culturing for 12-24 hours at the temperature of 37 ℃;
b. first-class seed liquid
Picking and inoculating single colonies from the flat plate which is subjected to the step a, inoculating the single colonies into a liquid TYS culture medium, and carrying out shake culture at 37 ℃ and 200rpm for 10-12h to obtain a primary seed solution;
c. second-stage seed liquid
And (3) taking the primary seed solution, inoculating the primary seed solution into a liquid TYS culture medium according to the inoculation amount of 1%, adding 10g/L of acetic acid or 10g/L of butyric acid as a carbon source, and performing shaking culture at 37 ℃ and 200rpm for 10-12h to obtain a secondary seed solution.
In the invention, the polyhydroxyalkanoate can be poly-3-hydroxybutyrate, 3-hydroxybutyrate and 3-hydroxyvalerate copolyester or 3-hydroxybutyrate and 4-hydroxybutyrate copolyester; the 3-hydroxybutyric acid and 3-hydroxyvaleric acid copolyester is an ester obtained by copolymerizing 3-hydroxybutyric acid and 3-hydroxyvaleric acid; the 3-hydroxybutyric acid and 4-hydroxybutyric acid copolymer ester is an ester obtained by copolymerizing 3-hydroxybutyric acid and 4-hydroxybutyric acid.
The Neptunomonas conchasum is separated from a marine environment and can grow well under the condition that the NaCl concentration is 25-50 g/L. Unlike the lower optimal growth temperature of common marine microorganisms, n. concharum JCM17730 is able to grow well at 30-37 ℃ of common industrial fermentations. Different from common fermentation industrial strains such as escherichia coli, lactic acid bacteria, corynebacterium glutamicum, saccharomyces cerevisiae and the like, the N.concharum JCM17730 has weak utilization degree of carbohydrate carbon sources such as glucose, fructose, sucrose and the like, but can utilize volatile fatty acids such as acetic acid, propionic acid, butyric acid and the like as carbon sources for rapid growth.
In the invention, the hyphosporon with the production capacity of the polyhydroxyalkanoate can utilize acetic acid or butyric acid as a carbon source, and accumulate a large amount of polyhydroxyalkanoate under the open condition without sterilization, wherein the proportion of polyester in the dry weight of bacteria can reach 69 percent at most, and the yield with the butyric acid as the carbon source can reach 40 percent at most of the theoretical yield. The method produces the polyhydroxyalkanoate by using two unusual carbon sources of acetic acid or butyric 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.
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FIG. 1 shows the fermenter culture for producing polyhydroxyalkanoate from butyric acid by using Hibiscus marinus.
Detailed Description
The present invention will be further illustrated with reference to the following specific examples, but the present invention is not limited to the following examples. The method is a conventional method unless otherwise specified. The starting materials are commercially available from the open literature 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, with the product designation poly (3-hydroxybutyrate-co-3-hydroxyvalerate) and a poly-3-hydroxybutyrate content of 88 mol%. The Neptomonas conchasum used in the following examples was purchased from the Japanese Collection of Microorganisms, and the representative strain was deposited in the Japanese Collection of Microorganisms under the accession number JCM 17730.
The method for preparing the seed liquid in the examples is as follows:
a. strain activation
Taking a strain glycerin tube stored in a refrigerator at the temperature of-80 ℃, streaking and inoculating the strain glycerin tube to a TYS culture medium plate, and culturing for 12-24 hours at the temperature of 37 ℃;
b. first-class seed liquid
Picking and inoculating single colonies from the flat plate which is subjected to the step a, inoculating the single colonies into a liquid TYS culture medium, and carrying out shake culture at 37 ℃ and 200rpm for 10-12h to obtain a primary seed solution;
c. second-stage seed liquid
And (3) taking the primary seed solution, inoculating the primary seed solution into a liquid TYS culture medium according to the inoculation amount of 1%, adding 10g/L of acetic acid or 10g/L of butyric acid as a carbon source, and performing shaking culture at 37 ℃ and 200rpm for 10-12h to obtain a secondary seed solution.
Method for freeze drying in the examples:
after acetic acid or butyric acid is used as a carbon source for fermentation, 30ml of fermentation liquor is taken, centrifuged at 8000rpm for 10min, supernatant is discarded, then deionized water is used for resuspending thalli, washing is carried out, centrifugation is carried out at 8000rpm for 10min again, thalli are collected, and freeze drying is carried out (a centrifugal tube filled with washed thalli precipitates is placed at minus 20 ℃ for freezing for 1h, and then is placed into a freeze vacuum drying instrument for freeze drying for 8-12h), so as to obtain a freeze-dried product;
in the examples the dry cell weight is measured as dry cell weight per liter of fermentation broth. The unit of cell dry weight is g/L. Dry cell weight (CDW for short) is (weight of centrifugal tube after freeze drying-weight of original air centrifugal tube) ÷ 0.03; the weight of the centrifugal tube after freeze drying and the weight of the original hollow centrifugal tube are both g; 0.03 represents taking a volume of the fermentation broth of 0.03L.
In the examples, the content of polyhydroxyalkanoate in the bacterial body is detected by the following method: the product was freeze-dried for esterification and then the monomer content was determined by gas chromatography.
Esterification reaction: putting 50mg of freeze-dried product into an esterification tube, adding 2mL of chloroform and 2mL of esterification solution (obtained by adding 15mL of concentrated sulfuric acid and 1g of benzoic acid into 500mL of methanol), mixing, covering, sealing, and esterifying at 100 deg.C for 4 hr; 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.
20mg of poly (3-hydroxybutyric acid-co-3-hydroxypentanoic acid) was taken and subjected to esterification reaction in the same manner as above to obtain a standard sample.
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.
During gas chromatography detection, a freeze-dried cell sample is compared with a poly (3-hydroxybutyrate-co-3-hydroxyvalerate) standard, and the freeze-dried cell sample contains a peak at the position of 3-hydroxybutyrate in the standard. Meanwhile, adding a freeze-dried cell sample into a chloroform solution, keeping the freeze-dried cell sample in a baking oven at 100 ℃ for 4 hours, cooling, taking a chloroform phase, adding ethanol with 6 times of volume, and separating out a white precipitate; and taking the precipitated precipitate, and performing esterification reaction and gas chromatography detection by adopting the steps, wherein the peak position is the same as that of the 3-hydroxybutyric acid in the standard substance. This indicates that the polyhydroxyalkanoate accumulated in the cells was poly-3-hydroxybutyrate.
Polymer content is defined as the ratio of polymer to dry cell weight, polymer yield ═ polymer content × dry cell weight.
In the following examples, butyric acid was quantitatively detected by high performance liquid chromatography as follows.
The specific conditions are as follows:
the instrument comprises the following steps: the Shimadzu corporation Essentia LC series HPLC instrument is equipped with a DGU-20A degasser, an LC-16 liquid pump, an SIL-16 type autosampler and an RID-20A detector.
Chromatographic conditions are as follows: Bio-Rad
HPX-87H (7.8X 300 mm); the flow rate is 0.50 mL/min; the column temperature is 55 ℃; the mobile phase was 7mM aqueous sulfuric acid.
The detection method comprises the following steps:
taking 0, 1, 2, 3, 4 and 5g/L butyric acid standard aqueous solution (Sigma-Aldrich, product number B103500), filtering with a 0.22 mu m microporous filter membrane, injecting 10 mu L of sample, carrying out HPLC detection, and drawing a standard curve by taking the chromatographic peak areas of butyric acid standard solutions with different concentrations as vertical coordinates and different concentrations as horizontal coordinates.
Centrifuging 2mL fermentation liquid at 12000rpm for 10min, transferring the fermentation supernatant into a new centrifuge tube, filtering with 0.22 μm microporous membrane, injecting 10 μ L, and performing HPLC detection.
And substituting the butyric acid chromatographic peak area of the fermentation supernatant of the sample to be detected into the standard curve, and calculating to obtain the butyric acid content of the fermentation supernatant of the sample to be detected.
In the following examples, the OD of the system containing Neurospora at the initial stage of fermentation600The values were all 0.1.
Example 1 production of polyhydroxyalkanoates by Neurospora with acetic acid (Shake flask culture)
Production of polyhydroxyalkanoates by using acetic acid from hyphosporon
1. Sterile preparation of sea-god monad seed liquid
(1) Strain activation
Taking strain glycerine tube stored in refrigerator at-80 deg.C, streaking and inoculating to TYS culture medium plate, and culturing at 37 deg.C for 12-24 hr.
(2) First class seed
Picking single colony from the plate completing the step (1), inoculating in liquid TYS culture medium, and culturing at 37 deg.C and 200rpm for 10-12h with shaking.
(3) Second grade seed
Inoculating the primary seed liquid obtained in the step (2) into a liquid TYS culture medium according to the inoculation amount of 1%, adding 10g/L of acetic acid as a carbon source, and performing shake culture at 37 ℃ and 200rpm for 10-12 h.
2. A liquid TYS medium was prepared, and acetic acid was added to a final concentration of 20g/L to adjust the pH to 7.
The following controls were also set up following the same procedure as above:
acetic acid was replaced with propionic acid and the concentration was replaced with 10 g/L;
3. inoculating the seed solution obtained in the step 1(3) into a liquid TYS culture medium containing 20g/L acetic acid according to an inoculation amount of 8% (namely 4ml of secondary seed solution and 46ml of culture medium), using a 250ml shake flask, wherein the total liquid loading is 50ml, and culturing at 37 ℃ and 200rpm for 40-48h to prepare a fermentation liquid.
The following controls were also set up following the same procedure as above:
acetic acid was replaced with propionic acid and the concentration was replaced with 10 g/L;
4. and (3) placing 30ml of fermentation liquor into a centrifugal tube with the volume of 50ml, centrifuging for 10min at 8000rpm, discarding the supernatant, then resuspending and cleaning the thalli by using deionized water, centrifuging for 10min at 8000rpm again, discarding the supernatant, placing the thalli at-20 ℃ for 1h, and then carrying out vacuum freeze drying for 8-12h to obtain the freeze-dried thalli. 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.
5. Transferring the freeze-dried thallus into an esterification tube with known mass, weighing the dry weight of the transferred thallus, adding 2mL of esterification solution (concentrated sulfuric acid is dissolved in methanol according to the volume percentage content of 3% to obtain sulfuric acid-methanol solution, adding benzoic acid serving as an internal standard into the sulfuric acid-methanol solution according to the final concentration of 1g/L to obtain esterification solution), 2mL of chloroform, covering and sealing, and reacting in an oven at 100 ℃ for 4 hours; cooling to room temperature, adding 1mL of deionized water, fully oscillating, standing and layering; after the chloroform phase and the water phase are completely separated, taking the chloroform phase and carrying out gas chromatography analysis;
a microsyringe from Agilent was used in an amount of 1. mu.L, and the polymer was quantitatively analyzed by an internal standard method and quantified based on the peak area.
Polymer content is defined as the ratio of polymer to dry cell weight, polymer yield ═ polymer content × dry cell weight.
By calculation, when acetic acid is obtained as a substrate, the dry weight of the cells is 3.95g/L, the polyhydroxyalkanoate accumulated in the cells is poly-3-hydroxybutyrate, and the yield is 1.62 g/L.
As a control, when propionic acid was used as a substrate, the dry cell weight was 1.96g/L, and no polyester was produced.
Open culture without inoculation of TYS acetic acid medium
1. A liquid TYS medium was prepared, and acetic acid was added to a final concentration of 20g/L to adjust the pH to 7. Without any microorganism inoculation, a 250ml shake flask is used, the total liquid loading is 50ml, the culture is carried out at 37 ℃ and 200rpm for 40-48h, and the fermentation liquid is prepared.
2. Since no inoculation is carried out, but the mixed bacteria grow under the open condition, the dry cell weight and the yield of the polyhydroxyalkanoate of the mixed bacteria culture are detected according to the method in the first step.
As a result, it was found that the cell dry weight was 0.17g/L, and accumulation of polyhydroxyalkanoate was not detected. This indicates that, with 20g/L of acetic acid as a carbon source, microorganisms rarely grow and the biomass of miscellaneous bacteria is low.
Shaking culture experiment of sea-god monad under acetic acid-free condition
1. According to the method of the one mentioned above, the medium for fermentation was replaced with a TYS medium without acetic acid addition (differing from TYS acetic acid medium only in that the components do not contain acetic acid), and the other steps were not changed.
2. The accumulation of the dry cell weight and the polyhydroxyalkanoate is detected, and the result shows that the dry cell weight is 1.12g/L after the hyphosporon is cultured in a shake flask under the condition of no acetic acid, and the accumulation of the polyhydroxyalkanoate is not detected.
It can be seen that the production of polyhydroxyalkanoates by hyphostroma is not sufficiently ensured by peptone and yeast extract in TYS medium alone, and that there should be growth of other bacteria by fermentation without sterilization.
Example 2 production of polyhydroxyalkanoates by Hibiscus using butyric acid (Shake flask culture)
Production of polyhydroxyalkanoate by using hyphosporon using butyric acid
1. Sterile preparation of sea-god monad seed liquid
(1) Strain activation
Taking strain glycerine tube stored in refrigerator at-80 deg.C, streaking and inoculating to TYS culture medium plate, and culturing at 37 deg.C for 12-24 hr.
(2) First class seed
Picking single colony from the plate completing the step (1), inoculating in liquid TYS culture medium, and culturing at 37 deg.C and 200rpm for 10-12h with shaking.
(3) Second grade seed
Inoculating the primary seed liquid obtained in the step (2) into a liquid TYS culture medium according to the inoculation amount of 1%, adding 10g/L butyric acid as a carbon source, and performing shake culture at 37 ℃ and 200rpm for 10-12 h.
2. Preparing a liquid TYS culture medium, adding butyric acid to a final concentration of 10g/L, and adjusting the pH value to 7.
3. Inoculating the seed solution obtained in the step 1(3) into a liquid TYS culture medium containing butyric acid according to the inoculation amount of 8%, using 250ml of shake flasks, wherein the total liquid loading is 50ml, and culturing at 37 ℃ and 200rpm for 40-48h to prepare a fermentation liquid.
4. And (3) placing 30ml of fermentation liquor into a centrifugal tube with the volume of 50ml, centrifuging for 10min at 8000rpm, discarding the supernatant, then resuspending and cleaning the thalli by using deionized water, centrifuging for 10min at 8000rpm again, discarding the supernatant, placing the thalli at-20 ℃ for 1h, and then carrying out vacuum freeze drying for 8-12h to obtain the freeze-dried thalli. 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.
5. Transferring the freeze-dried thallus into an esterification tube with known mass, weighing the dry weight of the transferred thallus, adding 2mL of esterification solution (concentrated sulfuric acid is dissolved in methanol according to the volume percentage content of 3% to obtain sulfuric acid-methanol solution, adding benzoic acid serving as an internal standard into the sulfuric acid-methanol solution according to the final concentration of 1g/L to obtain esterification solution), 2mL of chloroform, covering and sealing, and reacting in an oven at 100 ℃ for 4 hours; cooling to room temperature, adding 1mL of deionized water, fully oscillating, standing and layering; after the chloroform phase is completely separated from the water phase, taking the chloroform phase and carrying out gas chromatography analysis;
a microsyringe from Agilent was used in an amount of 1. mu.L, and the polymer was quantitatively analyzed by an internal standard method and quantified based on the peak area.
Polymer content is defined as the ratio of polymer to dry cell weight, polymer yield ═ polymer content × dry cell weight.
According to calculation, when butyric acid is obtained as a substrate, the dry weight of the cell is 5.01g/L, the polyhydroxyalkanoate accumulated in the cell is poly-3-hydroxybutyrate, and the yield is 3.49g/L and accounts for 69 percent of the dry weight of the cell. It can be seen that butyric acid, relative to acetic acid, enables higher dry cell weight and polyhydroxyalkanoate production. The theoretical highest yield of the poly-3-hydroxybutyrate synthesized by using the butyric acid as the carbon source is 0.98g of poly-3-hydroxybutyrate/g of butyric acid, and the actual yield obtained in the embodiment reaches 40% of the theoretical yield.
Open culture without inoculation of TYS butyric acid culture medium
1. Preparing a liquid TYS culture medium, adding butyric acid to a final concentration of 20g/L, and adjusting the pH value to 7. Without any microorganism inoculation, a 250ml shake flask is used, the total liquid loading is 50ml, the culture is carried out at 37 ℃ and 200rpm for 40-48h, and the fermentation liquid is prepared.
2. Since no inoculation is carried out, but the mixed bacteria grow under the open condition, the dry cell weight and the yield of the polyhydroxyalkanoate of the mixed bacteria culture are detected according to the method in the first step.
As a result, it was found that the cell dry weight was 0.06g/L, and accumulation of polyhydroxyalkanoate was not detected. Therefore, the 10g/L butyric acid is taken as a carbon source, so that the general microorganism rarely grows, and the biomass of the mixed bacteria is low.
Example 3 production of polyhydroxyalkanoate from butyric acid by Hibiscus
Production of polyhydroxyalkanoate by using hyphosporon using butyric acid
1. Sterile preparation of sea-god monad seed liquid
(1) Strain activation
Taking strain glycerine tube stored in refrigerator at-80 deg.C, streaking and inoculating to TYS culture medium plate, and culturing at 37 deg.C for 12-24 hr.
(2) First class seed
Picking single colony from the plate completing the step (1), inoculating in liquid TYS culture medium, and culturing at 37 deg.C and 200rpm for 10-12h with shaking.
(3) Second grade seed
Inoculating the primary seed liquid obtained in the step (2) into a liquid TYS culture medium according to the inoculation amount of 1%, adding 10g/L butyric acid as a carbon source, and performing shake culture at 37 ℃ and 200rpm for 10-12 h. The fermentation tank culture required secondary seed liquid total 300 ml.
2. 2700ml of TYS medium was prepared and butyric acid was added to a final concentration of 10 g/L. The medium was added to a 5L fermenter (Bailun Bio, model BLBIO-5 GJ-2).
3. Adding 300ml of secondary seed liquid into a fermentation tank, keeping the fermentation system at 3L, the fermentation temperature at 37 ℃, keeping the dissolved oxygen at 30% by correlating the stirring speed, adjusting the pH value by using 6M sodium hydroxide and 3M hydrochloric acid at 800rpm, and continuously fermenting and culturing under the condition that the pH value is 8, wherein the ventilation volume of air is 3L/min. Sampling 50ml of the remaining sample every 4 hours for detecting the dry cell weight and the yield of the polyhydroxyalkanoate, simultaneously detecting the butyric acid amount in real time, supplementing the butyric acid, and fermenting for 52 hours.
5. And (3) placing 30ml of fermentation liquor into a centrifugal tube with the volume of 50ml, centrifuging for 10min at 8000rpm, discarding the supernatant, then resuspending and cleaning the thalli by using deionized water, centrifuging for 10min at 8000rpm again, discarding the supernatant, placing the thalli at-20 ℃ for 1h, and then carrying out vacuum freeze drying for 8-12h to obtain the freeze-dried thalli. 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.
6. Transferring the freeze-dried thallus into an esterification tube with known mass, weighing the dry weight of the transferred thallus, adding 2mL of esterification solution (concentrated sulfuric acid is dissolved in methanol according to the volume percentage content of 3% to obtain sulfuric acid-methanol solution, adding benzoic acid serving as an internal standard into the sulfuric acid-methanol solution according to the final concentration of 1g/L to obtain esterification solution), 2mL of chloroform, covering and sealing, and reacting in an oven at 100 ℃ for 4 hours; 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;
7. 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.
8. Polymer content is defined as the ratio of polymer to dry cell weight, polymer yield ═ polymer content × dry cell weight.
The results show (figure 1), fermentation time is 48 hours, the yield of cell dry weight and polyhydroxyalkanoate reaches the highest, the cell dry weight is 35.07g/L, the polyester accumulated in cells is poly-3-hydroxybutyrate, the yield is 15.51g/L, the total amount of butyric acid consumed by fermentation is 54.17g/L, and the fermentation yield is 0.29g of poly-3-hydroxybutyrate/g of butyric acid. The theoretical highest yield of the poly-3-hydroxybutyrate synthesized by using the butyric acid as the carbon source is 0.98g of poly-3-hydroxybutyrate/g of butyric acid, and the actual yield obtained in the embodiment reaches 30% of the theoretical yield.
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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| CN105586304A (en) * | 2014-11-18 | 2016-05-18 | 北京化工大学 | Recombinant strain for producing glycolate based polymers and application of recombinant strain |
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| CN105586304A (en) * | 2014-11-18 | 2016-05-18 | 北京化工大学 | Recombinant strain for producing glycolate based polymers and application of recombinant strain |
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