CN111100901B - Method for producing 1, 3-propylene glycol by glycerol fermentation - Google Patents
Method for producing 1, 3-propylene glycol by glycerol fermentation Download PDFInfo
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- CN111100901B CN111100901B CN201811255629.5A CN201811255629A CN111100901B CN 111100901 B CN111100901 B CN 111100901B CN 201811255629 A CN201811255629 A CN 201811255629A CN 111100901 B CN111100901 B CN 111100901B
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- 238000000855 fermentation Methods 0.000 title claims abstract description 135
- 230000004151 fermentation Effects 0.000 title claims abstract description 132
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 title claims abstract description 99
- YPFDHNVEDLHUCE-UHFFFAOYSA-N propane-1,3-diol Chemical compound OCCCO YPFDHNVEDLHUCE-UHFFFAOYSA-N 0.000 title claims abstract description 67
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- DNIAPMSPPWPWGF-VKHMYHEASA-N (+)-propylene glycol Chemical compound C[C@H](O)CO DNIAPMSPPWPWGF-VKHMYHEASA-N 0.000 description 11
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- PKAUICCNAWQPAU-UHFFFAOYSA-N 2-(4-chloro-2-methylphenoxy)acetic acid;n-methylmethanamine Chemical compound CNC.CC1=CC(Cl)=CC=C1OCC(O)=O PKAUICCNAWQPAU-UHFFFAOYSA-N 0.000 description 1
- 241000180579 Arca Species 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
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- 230000015572 biosynthetic process Effects 0.000 description 1
- KDYFGRWQOYBRFD-NUQCWPJISA-N butanedioic acid Chemical compound O[14C](=O)CC[14C](O)=O KDYFGRWQOYBRFD-NUQCWPJISA-N 0.000 description 1
- AXCZMVOFGPJBDE-UHFFFAOYSA-L calcium dihydroxide Chemical compound [OH-].[OH-].[Ca+2] AXCZMVOFGPJBDE-UHFFFAOYSA-L 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P39/00—Processes involving microorganisms of different genera in the same process, simultaneously
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
- C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
- C12P7/18—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic polyhydric
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Abstract
The invention relates to a method for producing 1, 3-propylene glycol by glycerol fermentation, which comprises the following steps of firstly, culturing a fermentation strain under the micro-aerobic condition to obtain a fermentation seed solution; then carrying out microalgae culture under an autotrophic condition to obtain a microalgae seed solution; then inoculating fermentation seed liquid into a fermentation culture medium for micro-aerobic fermentation, adding the micro-algae seed liquid when the acetic acid concentration is more than 3g/L after fermentation, and culturing until the fermentation is finished; and finally, adjusting the pH value to be alkaline, standing and layering to obtain a fermentation clear liquid containing the 1, 3-propylene glycol. The method of the invention utilizes the combination of microalgae cell metabolism and a fermentation system to strengthen the oxidative phosphorylation of the substrate level in the glycerol metabolism and improve the fermentation level.
Description
Technical Field
The invention belongs to the technical field of bioengineering, and particularly relates to a method for producing 1, 3-propylene glycol by glycerol fermentation.
Background
1, 3-Propanediol (1, 3-Propanediol, PDO) is mainly used for producing a novel polyester PTT (polytrimethylene terephthalate) with excellent performance. The polyester synthesized by the method has unique properties and excellent performance, and can enable the polyester plastic to have the biodegradable characteristic of easy natural circulation. In recent years, as important organic synthesis raw materials and intermediates, research and development are hot due to unique properties and wide application.
The industrial production method of 1, 3-propylene glycol mainly comprises two main types of chemical methods and biological methods. Biological methods are the hot spot of current research and can be divided into a glucose one-step conversion method and a glycerol conversion method according to different raw materials. Although biological methods began earlier, they did not gradually attract attention until the eighties of the twentieth century. Compared with a chemical method, the microbial conversion method has the characteristics of mild conditions, simple and convenient operation, good selectivity, energy conservation, less equipment investment, good environment and the like, is a method with the lowest production cost and the least pollution, and meets the requirements of current 'green chemical industry' and 'sustainable development'.
At present, glycerol is taken as a substrate, and a technical route for preparing 1, 3-propylene glycol by virtue of Klebsiella bioconversion is widely adopted due to simple metabolic pathway and easy modification of fermentation strains. The Klebsiella is a typical facultative anaerobic microorganism which can grow under anaerobic, microaerobic and aerobic conditions, so that the process for preparing the 1, 3-propylene glycol by bioconversion of the glycerol is mainly divided into an aerobic mode, a microaerobic mode and an anaerobic mode. Chinese patent CN1348007A discloses a method for producing 1, 3-propanediol by microbial micro-aerobic fermentation, wherein microbial cells used in the method can convert glycerol into 1, 3-propanediol not only under anaerobic conditions but also under micro-aerobic conditions. In recent years, starting from the metabolic pathway of the Klebsiella, the aerobic, micro-aerobic and anaerobic modes are deeply researched, and various technical schemes are formed. Chinese patent CN1434122A discloses a method for producing 1, 3-propanediol by two-stage double-substrate integrated fermentation, wherein the second-stage seed culture is carried out by taking glucose and glycerol as mixed double substrates and integrating the second-stage seed culture under aerobic condition and the anaerobic transformation of the glycerol under anaerobic condition in the same fermentation tank. Chinese patent CN101307335A discloses an improved method for producing 1, 3-propylene glycol by glycerol anaerobic fermentation, which is characterized in that alkaline calcium salt is added in the fermentation process to realize the online regulation and control of pH value, and the metabolism of glycerol between two branches of reduction and oxidation is improved, thereby being beneficial to the growth of thalli and the production of products. Chinese patent CN102864177A discloses a method for promoting microbial fermentation to produce 1, 3-propylene glycol, the fermentation process is divided into micro-aerobic fermentation and anaerobic fermentation in turn, when the OD value is more than 7, the micro-aerobic fermentation is converted into the anaerobic fermentation. The method can effectively promote the conversion of the glycerol and greatly improve the concentration of the 1, 3-propylene glycol in the final product.
With the development of biotechnology, the idea of adapting fermentation process by strain modification by using genetic engineering technical means has also been realized. Chinese patent CN 106399217A discloses construction and application of a Klebsiella bacteria arcA gene deletion strain, and is characterized in that a homologous recombination method is utilized to knock out a key gene arcA for producing 1, 3-propanediol Klebsiella to inhibit TCA cycle, so that TCA cycle activity of cells under a micro-aerobic condition is improved, regeneration of reducing power is enhanced, and yield of 1, 3-propanediol is improved. Chinese patent CN102199570A discloses a method for constructing genetically engineered bacteria to enhance the production of 1, 3-propanediol by microorganisms, which comprises constructing an expression vector inserted with malic enzyme gene; transferring the expression vector inserted with the malic enzyme gene into a host bacterium for producing 1, 3-propanediol; adding an inducer to induce the excessive expression of the malic enzyme gene in the fermentation culture process; the 1, 3-propylene glycol is produced by adopting an aerobic fermentation mode and adding substrate glycerol.
Although the methods start from the facultative anaerobic characteristic of the Klebsiella and achieve certain effects through methods such as stage regulation, strain transformation and the like, the methods are discovered from the research of metabolic pathway mechanisms: (1) the selectivity of glycerol is highest under an anaerobic condition, but high-flux nitrogen needs to be introduced in the process to maintain a micro-aerobic condition, so that the energy consumption is high; (2) under the micro-aerobic and aerobic conditions, a lot of fermentation byproducts exist, and currently, genetic engineering bacteria are mostly adopted, but the fermentation level is not high.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a method for producing 1, 3-propylene glycol by glycerol fermentation. The method of the invention utilizes the combination of microalgae cell metabolism and a fermentation system to strengthen the oxidative phosphorylation of the substrate level in the glycerol metabolism and improve the fermentation level.
The invention relates to a method for producing 1, 3-propylene glycol by glycerol fermentation, which comprises the following steps:
(1) culturing fermentation strains: culturing a fermentation strain by using a seed culture medium under the micro-aerobic condition to obtain a fermentation seed solution;
(2) culturing microalgae: culturing microalgae under autotrophic condition to obtain microalgae seed solution;
(3) and (3) fermentation process: inoculating fermentation seed liquid into a fermentation culture medium, fermenting under the condition of micro-oxygen, adding the microalgae seed liquid when the acetic acid concentration is more than 3g/L, and culturing until the fermentation is finished;
(4) adjusting the pH value of the terminated fermentation liquor to be alkaline, standing and layering to obtain a fermentation clear liquid containing the 1, 3-propylene glycol.
In the invention, the fermentation bacteria in the step (1) are microorganisms capable of producing 1, 3-propanediol by glycerol fermentation, mainly facultative anaerobes, such as Klebsiella (Klebsiella)Klebsiella pneumoniae) Citrobacter freundii: (Citrobacter freundii）Etc., preferably Klebsiella.
In the invention, the activated zymophyte is inoculated into a seed culture medium in the step (1), and the inoculation volume ratio is 3-10%, preferably 5-10%; the culture temperature is 25-40 ℃, and preferably 35-40 ℃; the pH value is 6-9, preferably 6.5-7.2. The micro-oxygen condition adopts a mode of introducing low air volume, and the air volume is less than 0.1vvm, preferably 0.02-0.08 vvm.
In the present invention, the microalgae in step (2) is derived from microalgae having photoautotrophic and mixotrophic activities, i.e. the microalgae has the characteristics of using photoautotrophic growth and using organic carbon source for growth, such as green algae (Chlorella vulgaris, for example) can be selectedChlorella) Chlamydomonas (a)Chlamydomonas) Scenedesmus (Scenedesmus)Scenedesmus) Fibroalgae(Ankistrodesmus) And the like.
In the present invention, as the culture medium for the autotrophic culture in step (2), a liquid culture medium for culturing microalgae, such as BG11, SE, and BBM, which are well known in the art, may be used, and BG11 is preferable. The autotrophic culture conditions are as follows: the illumination intensity is 2000-7000 Lux, the light-dark time ratio is 7: 5-2: 1, the pH value is 7-9, the temperature is 25-40 ℃, and CO is introduced during ventilation2The content is 3-10 v%. Culturing until the dry weight of the algae cells in the microalgae seed solution reaches 5 g/L-20 g/L.
In the invention, the inoculation volume ratio of the fermentation seed liquid in the step (3) is 2-20%, preferably 10-15%. The micro-oxygen condition is as follows: the air ventilation is less than 0.1vvm, preferably 0 to 0.05. The fermentation conditions were: the fermentation temperature is 30-42 ℃, and preferably 35-40 ℃; the stirring speed is 100-500 rpm, preferably 200-400 rpm; the pH is controlled to be 6 to 7.5, preferably 6.8 to 7.2.
In the invention, the volume ratio of the microalgae seed liquid in the step (3) is 2-20%, preferably 5-10%. After the microalgae seed liquid is added, the pH is controlled to be 7.5-8.5, and preferably 7.5-8.
In the invention, the glycerol is fed during the fermentation process in the step (3) to control the concentration of the glycerol in the fermentation system to be 20 g/L-40 g/L.
In the invention, after the fermentation in the step (3) is finished, the pH value of the fermentation liquid is adjusted to 8.5-10, preferably 8.5-9.5. The pH value can be adjusted by adopting liquid strong base with the mass concentration of 30-40%, wherein the strong base is sodium hydroxide, calcium hydroxide, potassium hydroxide and the like, and the sodium hydroxide is preferred.
Compared with the prior art, the method has the following beneficial effects:
(1) according to the invention, the metabolic characteristics of microalgae and the micro-aerobic fermentation metabolic pathway for preparing 1, 3-propylene glycol by glycerol conversion are organically combined, the problem of cytotoxicity of polar molecules generated by dissolving carbon dioxide in a fermentation system to fermentation thalli is solved, and meanwhile, the endogenous consumption of a fermentation by-product acetic acid reduces the ion concentration in the fermentation system, thereby being beneficial to prolonging the fermentation period and improving the fermentation level. And the combination of the two can increase the pH value of the fermentation system, thereby reducing the dosage of the pH regulator.
(2) The microalgae seed solution is inoculated when the acetic acid concentration is more than 3g/L after fermentation, which is beneficial to the horizontal oxidative phosphorylation of the substrate in the glycerol metabolism, provides more ATP and reducing power for the fermentation thalli, and is beneficial to the synthesis of 1, 3-propanediol.
(3) According to the invention, the pH is controlled to be 6-7.5 in the fermentation process, the pH is controlled to be 7.5-8.5 after the microalgae is added, and the pH is adjusted to be 8.5-10 after the fermentation is finished, so that the fermentation of microalgae cells in a fermentation system is facilitated, the pH is increased, the separation of the thallus cells after the fermentation is finished is facilitated, and the fermentation clear liquid is easily obtained, thereby reducing the separation process and improving the production efficiency.
(4) The method has the advantages of high production efficiency, low energy consumption and good industrial application prospect.
Detailed Description
The method and effects of the present invention will be described in further detail by examples. The embodiments are implemented on the premise of the technical scheme of the invention, and detailed implementation modes and specific operation processes are given, but the protection scope of the invention is not limited by the following embodiments.
The experimental procedures in the following examples are, unless otherwise specified, conventional in the art. The experimental materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified.
In the embodiment of the invention, a liquid phase analysis system is formed by a Waters 2695 separation system and a Waters 2414 differential detector, wherein an Aminex HPX-87H organic acid and alcohol analysis column is selected as a separation column for separating acids and alcohols. And establishing a standard map by using succinic acid, lactic acid, glycerol, acetic acid, 1, 3-propylene glycol and ethanol standard samples, and analyzing products in the reaction process.
In the examples of the present invention, OD was measured with a spectrophotometer600、OD280Respectively representing the content of solid matters and the content of protein macromolecules in the fermentation liquor.
In the embodiment of the invention, the used strain is the published Klebsiella pneumoniae (C.) (Klebsiella pneumoniae) The strain is from China petrochemical industry smoothing petrochemical research institute patent strains, is preserved in China general microbiological culture center of Committee for culture Collection of microorganisms, and has the preservation number: CGMCC number 0798.
In the examples of the present invention, the following Cellulosite (A) is usedAnkistrodesmus sp.) SS-B7, which has been deposited in China general microbiological culture Collection center of China Committee for culture Collection of microorganisms (CGMCC) No. 7478 in 2013, 4 months and 15 days, and the deposition address is the institute of microbiology, China academy of sciences No. 3, North West Lu No. 1 Hospital, Chaoyang, Beijing. The strain has been disclosed in CN 105713836AThe collection and proof of survival were submitted.
In the examples of the present invention, Chlorella (A) and (B) are usedChlorella sp.) SF-B1 has been deposited in the general microbiological center of China Committee for culture Collection of microorganisms (CGMCC) No. 11005 at 7/6/2015 with the collection address of the institute for microbiology, China academy of sciences No. 3, Siro 1, North Cheng, the sunward area, Beijing.
In the examples of the present invention, the basic composition of the Klebsiella seed medium and the fermentation medium are shown in Table 1.
TABLE 1 Klebsiella seed Medium and fermentation Medium
The culture of the microalgae adopts BG11 culture medium, and the formula is shown in tables 2 and 3.
TABLE 2 BG11 culture Medium
Table 3 composition of a5+ Co solution in table 1
Example 1
(1) And (3) strain culture: taking 70mL of activated Klebsiella bacterium liquid, mixing the activated Klebsiella bacterium liquid with 630mL of seed culture medium in a 1L fermentation tank, and carrying out microaerophilic culture with the ventilation volume of 0.02 vvm; the culture conditions were: culturing at 37 deg.C, stirring at 200rpm, controlling pH at 7.0, and culturing for 18 hr to obtain fermented seed solution.
(2) Culturing microalgae: mixing 60mL of Cellulomonas sp-B7 algae solution in logarithmic growth phase and 540mL of microalgae culture medium in column reactor (inner diameter of 40mm and height of 500 mm), introducing CO during culture2Air mixed gas with the content of 3v percent, the illumination intensity of 4000Lux, the culture temperature of 28 ℃, the pH value of 7.5,the illumination period is 24h, the light-dark time ratio is 14:10, and the microalgae seed liquid is cultured until the dry weight of the algae cells in the microalgae seed liquid reaches 18.2 g/L.
(3) And (3) fermentation process: the volume of the fermentation tank is 15L, and the liquid filling volume is 7L.
Taking 700mL of fermentation seed liquid, inoculating the fermentation seed liquid into 6.3L of fermentation medium, carrying out micro-aerobic fermentation, and feeding glycerol in the fermentation process to maintain the concentration of the glycerol in a fermentation system at 40 g/L; the culture temperature is controlled at 37 ℃, the pH value is 7.0 and the stirring speed is 400rpm in the fermentation process. Fermenting until the concentration of acetic acid in the fermentation liquor is 3.2g/L, and adding 350mL of microalgae seed liquid. The other control parameters are unchanged.
After fermenting for 40h, the concentration of 1, 3-propylene glycol is detected to be 75.64g/L, the concentration of acetic acid is detected to be 5.4g/L, and the conductivity is detected to be 15.2 mS/cm. Ending fermentation, adjusting pH to 8.5 with 30% sodium hydroxide, standing for 8 hr, layering the fermentation liquid, and collecting supernatant OD600、OD280The detection values were 1.1 and 12.4, respectively.
Example 2
(1) And (3) strain culture: taking 70mL of activated Klebsiella bacterium liquid, mixing the activated Klebsiella bacterium liquid with 630mL of seed culture medium in a 1L fermentation tank, and carrying out microaerophilic culture with the air introduction amount of 0.06 vvm; the culture conditions were: culturing at 37 deg.C, stirring at 300rpm, controlling pH at 7.2, and culturing for 18 hr to obtain fermented seed solution.
(2) Culturing microalgae: mixing 30mL of fibrophyta SS-B7 algae solution in logarithmic growth phase and 570mL of culture medium in column reactor (inner diameter of 40mm and height of 500 mm), introducing CO during culture2Air mixed gas with the content of 10v%, the illumination intensity of 7000Lux, the culture temperature of 37 ℃, the pH value of 7.5, the illumination period of 24 hours and the light-dark time ratio of 16:8 are cultured until the dry weight of the algae cells in the microalgae seed liquid reaches 19.6 g/L.
(3) And (3) fermentation process: the volume of the fermentation tank is 15L, and the liquid filling volume is 7L.
1050mL of fermentation seed liquid is taken and inoculated into 5.95L of fermentation medium for micro-aerobic fermentation, and glycerol is fed during the fermentation process so as to maintain the concentration of the glycerol in the fermentation system at 30 g/L; the fermentation process controlled the culture temperature at 37 deg.C, pH 7.2, and stirring speed at 300 rpm. Fermenting to a stable period, and adding 600mL of microalgae seed liquid. The other control parameters are unchanged.
After fermenting for 40h, the concentration of 1, 3-propylene glycol is detected to be 76.21g/L, the concentration of acetic acid is detected to be 3.2g/L, and the conductivity is detected to be 14.1 mS/cm. Ending fermentation, adjusting pH to 9 with 30% potassium hydroxide, standing for 8 hr, layering the fermentation liquid, and collecting supernatant OD600、OD280The detection values were 0.9 and 11.3, respectively.
Example 3
The fermentation process and process conditions were the same as in example 1. The difference lies in that: after the microalgae seed liquid is added in the fermentation process, the pH is regulated to 7.5.
After fermenting for 40h, the concentration of 1, 3-propylene glycol is detected to be 78.2g/L, the concentration of acetic acid is detected to be 4.6g/L, and the conductivity is detected to be 16.8 mS/cm. Ending fermentation, adjusting pH to 8.5, standing for 8 hr, layering the fermentation liquid, and collecting supernatant OD600、OD280The detection values were 1.0 and 10.6, respectively.
Example 4
The fermentation process and process conditions were the same as in example 2. The difference lies in that: after the microalgae seed liquid is added in the fermentation process, the pH value is regulated to 8.5.
After fermenting for 40h, the concentration of 1, 3-propylene glycol is detected to be 72.1g/L, the concentration of acetic acid is detected to be 2.8g/L, and the conductivity is detected to be 13.6 mS/cm. Ending fermentation, adjusting pH to 9, standing for 8 hr, layering the fermentation liquid, and collecting supernatant OD600、OD280The detection values were 0.8 and 10.4, respectively.
Example 5
The fermentation process and process conditions were the same as in example 1. The difference lies in that: the microalgae is Chlorella (Chlorella vulgaris)Chlorella sp.）SF-B1。
After fermenting for 40h, the concentration of 1, 3-propylene glycol is detected to be 76.37g/L, the concentration of acetic acid is detected to be 5.5g/L, and the conductivity is detected to be 15.1 mS/cm. Ending fermentation, adjusting pH to 8.5 with 30% sodium hydroxide, standing for 8 hr, layering the fermentation liquid, and collecting supernatant OD600、OD280The detection values were 1.2 and 12.3, respectively.
Example 6
The fermentation process and process conditions were the same as in example 1. The difference lies in that: and (3) in the fermentation process in the step (3), the micro-oxygen adopts a form of introducing air, and the air introduction amount is 0.05 vvm.
After fermenting for 40h, the concentration of 1, 3-propylene glycol is detected to be 76.14g/L, the concentration of acetic acid is detected to be 5.2g/L, and the conductivity is detected to be 14.6 mS/cm. Ending fermentation, adjusting pH to 8.5 with 30% sodium hydroxide, standing for 8 hr, layering the fermentation liquid, and collecting supernatant OD600、OD280The detection values are 1.2 and 12.6 respectively.
Comparative example 1
The fermentation process and process conditions were the same as in example 1. The difference lies in that the Phaeodactylum tricornutum which only has photoautotrophic property and does not have heterotrophic growth characteristic is selected (A)Phacodactylum tricornutum) Instead of Cellulosium sp-B7.
After fermenting for 40h, the detected concentration of 1, 3-propylene glycol is 56.32g/L, the concentration of acetic acid is 13.3g/L, and the conductivity is 27.7 mS/cm. Ending fermentation, adjusting pH to 8.5, standing for 8 hr, layering the fermentation liquid, and collecting supernatant OD600、OD280The detection values were 3.3 and 25.9, respectively.
Comparative example 2
The fermentation process and process conditions were the same as in example 1. The difference lies in that: according to the 'synchronous mixed culture' mode disclosed in the Chinese patent CN103103129A, the fermented seed liquid and the microalgae seed liquid are synchronously added into a fermentation system, and the rest process is completely the same as that of the embodiment 1. After fermenting for 40h, the concentration of 1, 3-propylene glycol is detected to be 55.26g/L, the concentration of acetic acid is detected to be 12.9g/L, and the conductivity is detected to be 27.2 mS/cm. Ending fermentation, adjusting pH to 8.5, standing for 8 hr, layering the fermentation liquid, and collecting supernatant OD600、OD280The detection values were 2.9 and 24.6, respectively.
Comparative example 3
The fermentation process and process conditions were the same as in example 1. The difference lies in that: and (4) after the fermentation in the step (3) is finished, not adjusting the pH of the fermentation liquor. Standing for 8h, layering the fermentation liquor, and collecting supernatant OD600、OD280The detection values were 3.3 and 25.9, respectively.
Claims (15)
1. A method for producing 1, 3-propylene glycol by glycerol fermentation is characterized by comprising the following steps:
(1) culturing fermentation strains: fermenting strain under micro-aerobic conditionObtaining a fermentation seed solution; the zymocyte is Klebsiella which can produce 1, 3-propylene glycol by glycerol fermentation (Klebsiella pneumoniae)Klebsiella pneumoniae）；
(2) Culturing microalgae: culturing microalgae under autotrophic condition to obtain microalgae seed solution; the microalgae is fibroalgae (A)Ankistrodesmus sp .) SS-B7 or Chlorella (Chlorella vulgaris)Chlorella sp.) SF-B1；
(3) And (3) fermentation process: inoculating fermentation seed liquid into a fermentation culture medium, fermenting under the condition of micro-oxygen, adding the microalgae seed liquid when the acetic acid concentration is more than 3g/L, and culturing until the fermentation is finished; in the fermentation process, glycerol is fed back to control the concentration of the glycerol in the fermentation system to be 20 g/L-40 g/L;
(4) adjusting the pH value of the terminated fermentation liquor to be alkaline, standing and layering to obtain a fermentation clear liquid containing the 1, 3-propylene glycol.
2. The method of claim 1, wherein: inoculating the activated zymophyte into a seed culture medium, wherein the inoculation volume ratio is 3-10%, the culture temperature is 25-40 ℃, and the pH value is 6-9.
3. The method of claim 2, wherein: the inoculation volume ratio is 5-10%, the culture temperature is 35-40 ℃, and the pH value is 6.5-7.2.
4. The method of claim 1, wherein: the aeration quantity adopted under the micro-aerobic condition in the step (1) is less than 0.1 vvm.
5. The method of claim 4, wherein: the ventilation volume is 0.02-0.08 vvm.
6. The method of claim 1, wherein: adopting BG11, SE or BBM as culture medium for culturing microalgae in the step (2), wherein the autotrophic culture condition is as follows: the illumination intensity is 2000-7000 Lux, the light-dark time ratio is 7: 5-2: 1, the pH value is 7-9,the temperature is 25-40 ℃, and CO is introduced in the air2The content is 3v% -10 v%; culturing until the dry weight of the algae cells in the microalgae seed solution reaches 5 g/L-20 g/L.
7. The method of claim 6, wherein: the culture medium adopts BG11 culture medium.
8. The method of claim 1, wherein: the inoculation volume ratio of the fermentation seed liquid in the step (3) is 2-20%, and the air ventilation is less than 0.1 vvm; the fermentation conditions were: the fermentation temperature is 30-42 ℃, the stirring speed is 100-500 rpm, and the pH is controlled to be 6-7.5.
9. The method of claim 8, wherein: the inoculation volume ratio of the fermentation seed liquid is 10-15%, and the air ventilation amount is 0-0.05 vvm; the fermentation conditions were: the fermentation temperature is 35-40 ℃, the stirring speed is 200-400 rpm, and the pH is controlled to be 6.8-7.2.
10. The method of claim 1, wherein: the adding volume ratio of the microalgae seed liquid in the step (3) is 2-20%.
11. The method of claim 10, wherein: the volume ratio of the microalgae seed liquid is 5-10%.
12. The method of claim 1, wherein: after the microalgae seed liquid is added, the pH is controlled to be 7.5-8.5.
13. The method of claim 12, wherein: the pH value is controlled to be 7.5-8.
14. The method of claim 1, wherein: and (4) after the fermentation in the step (3) is finished, adjusting the pH of the finished fermentation liquor to 8.5-10.
15. The method of claim 14, wherein: and adjusting the pH value of the terminated fermentation liquor to 8.5-9.5.
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