CN115369130A - Method for producing 1,3-propylene glycol through fermentation regulation and control based on cofactor - Google Patents
Method for producing 1,3-propylene glycol through fermentation regulation and control based on cofactor Download PDFInfo
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- CN115369130A CN115369130A CN202211187786.3A CN202211187786A CN115369130A CN 115369130 A CN115369130 A CN 115369130A CN 202211187786 A CN202211187786 A CN 202211187786A CN 115369130 A CN115369130 A CN 115369130A
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- 238000000855 fermentation Methods 0.000 title claims abstract description 85
- 230000004151 fermentation Effects 0.000 title claims abstract description 85
- YPFDHNVEDLHUCE-UHFFFAOYSA-N propane-1,3-diol Chemical compound OCCCO YPFDHNVEDLHUCE-UHFFFAOYSA-N 0.000 title claims abstract description 59
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 9
- 241000588747 Klebsiella pneumoniae Species 0.000 claims abstract description 28
- AUNGANRZJHBGPY-SCRDCRAPSA-N Riboflavin Chemical compound OC[C@@H](O)[C@@H](O)[C@@H](O)CN1C=2C=C(C)C(C)=CC=2N=C2C1=NC(=O)NC2=O AUNGANRZJHBGPY-SCRDCRAPSA-N 0.000 claims abstract description 28
- 241000863430 Shewanella Species 0.000 claims abstract description 25
- AUNGANRZJHBGPY-UHFFFAOYSA-N D-Lyxoflavin Natural products OCC(O)C(O)C(O)CN1C=2C=C(C)C(C)=CC=2N=C2C1=NC(=O)NC2=O AUNGANRZJHBGPY-UHFFFAOYSA-N 0.000 claims abstract description 14
- 229960002477 riboflavin Drugs 0.000 claims abstract description 14
- 235000019192 riboflavin Nutrition 0.000 claims abstract description 14
- 239000002151 riboflavin Substances 0.000 claims abstract description 14
- 239000001963 growth medium Substances 0.000 claims abstract description 12
- 230000002829 reductive effect Effects 0.000 claims abstract description 8
- PEDCQBHIVMGVHV-UHFFFAOYSA-N glycerol group Chemical group OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 87
- 239000002609 medium Substances 0.000 claims description 19
- 238000000034 method Methods 0.000 claims description 18
- DNIAPMSPPWPWGF-VKHMYHEASA-N (+)-propylene glycol Chemical compound C[C@H](O)CO DNIAPMSPPWPWGF-VKHMYHEASA-N 0.000 claims description 17
- 229920000166 polytrimethylene carbonate Polymers 0.000 claims description 17
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 9
- 229910052799 carbon Inorganic materials 0.000 claims description 9
- 239000007788 liquid Substances 0.000 claims description 9
- 238000011218 seed culture Methods 0.000 claims description 9
- 229910021591 Copper(I) chloride Inorganic materials 0.000 claims description 3
- OXBLHERUFWYNTN-UHFFFAOYSA-M copper(I) chloride Chemical compound [Cu]Cl OXBLHERUFWYNTN-UHFFFAOYSA-M 0.000 claims description 3
- 239000006227 byproduct Substances 0.000 abstract description 11
- 230000000694 effects Effects 0.000 abstract description 4
- 238000009776 industrial production Methods 0.000 abstract description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 16
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 15
- 239000000243 solution Substances 0.000 description 13
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 12
- KDYFGRWQOYBRFD-UHFFFAOYSA-N succinic acid Chemical compound OC(=O)CCC(O)=O KDYFGRWQOYBRFD-UHFFFAOYSA-N 0.000 description 8
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 6
- OWBTYPJTUOEWEK-UHFFFAOYSA-N butane-2,3-diol Chemical compound CC(O)C(C)O OWBTYPJTUOEWEK-UHFFFAOYSA-N 0.000 description 6
- 239000004310 lactic acid Substances 0.000 description 6
- 235000014655 lactic acid Nutrition 0.000 description 6
- 229930027945 nicotinamide-adenine dinucleotide Natural products 0.000 description 4
- BOPGDPNILDQYTO-NNYOXOHSSA-N nicotinamide-adenine dinucleotide Chemical compound C1=CCC(C(=O)N)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]2[C@H]([C@@H](O)[C@@H](O2)N2C3=NC=NC(N)=C3N=C2)O)O1 BOPGDPNILDQYTO-NNYOXOHSSA-N 0.000 description 4
- -1 polytrimethylene terephthalate Polymers 0.000 description 4
- 239000001384 succinic acid Substances 0.000 description 4
- 238000003786 synthesis reaction Methods 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- AKXKFZDCRYJKTF-UHFFFAOYSA-N 3-Hydroxypropionaldehyde Chemical compound OCCC=O AKXKFZDCRYJKTF-UHFFFAOYSA-N 0.000 description 3
- 238000002835 absorbance Methods 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- HGINCPLSRVDWNT-UHFFFAOYSA-N Acrolein Chemical compound C=CC=O HGINCPLSRVDWNT-UHFFFAOYSA-N 0.000 description 2
- 239000002028 Biomass Substances 0.000 description 2
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 2
- 241000588748 Klebsiella Species 0.000 description 2
- LCTONWCANYUPML-UHFFFAOYSA-M Pyruvate Chemical compound CC(=O)C([O-])=O LCTONWCANYUPML-UHFFFAOYSA-M 0.000 description 2
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 2
- 239000003225 biodiesel Substances 0.000 description 2
- 238000010170 biological method Methods 0.000 description 2
- 238000009903 catalytic hydrogenation reaction Methods 0.000 description 2
- 238000009472 formulation Methods 0.000 description 2
- 239000008103 glucose Substances 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 238000011081 inoculation Methods 0.000 description 2
- 238000004811 liquid chromatography Methods 0.000 description 2
- 230000000813 microbial effect Effects 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 229920000728 polyester Polymers 0.000 description 2
- 229920002215 polytrimethylene terephthalate Polymers 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 239000011734 sodium Substances 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 231100000331 toxic Toxicity 0.000 description 2
- 230000002588 toxic effect Effects 0.000 description 2
- AXPZIVKEZRHGAS-UHFFFAOYSA-N 3-benzyl-5-[(2-nitrophenoxy)methyl]oxolan-2-one Chemical compound [O-][N+](=O)C1=CC=CC=C1OCC1OC(=O)C(CC=2C=CC=CC=2)C1 AXPZIVKEZRHGAS-UHFFFAOYSA-N 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 241000588923 Citrobacter Species 0.000 description 1
- 241000193403 Clostridium Species 0.000 description 1
- 241000588914 Enterobacter Species 0.000 description 1
- IAYPIBMASNFSPL-UHFFFAOYSA-N Ethylene oxide Chemical compound C1CO1 IAYPIBMASNFSPL-UHFFFAOYSA-N 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- 241001223867 Shewanella oneidensis Species 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 229920000704 biodegradable plastic Polymers 0.000 description 1
- 239000003054 catalyst Substances 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- 239000002537 cosmetic Substances 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
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- 239000003814 drug Substances 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 150000002391 heterocyclic compounds Chemical class 0.000 description 1
- 230000036571 hydration Effects 0.000 description 1
- 238000006703 hydration reaction Methods 0.000 description 1
- 238000007037 hydroformylation reaction Methods 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 239000000543 intermediate Substances 0.000 description 1
- 238000011031 large-scale manufacturing process Methods 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000037353 metabolic pathway Effects 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 238000009629 microbiological culture Methods 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 239000000178 monomer Substances 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- 229920000570 polyether Polymers 0.000 description 1
- 229920002635 polyurethane Polymers 0.000 description 1
- 239000004814 polyurethane Substances 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 125000001452 riboflavin group Chemical group 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 235000013619 trace mineral Nutrition 0.000 description 1
- 239000011573 trace mineral Substances 0.000 description 1
- 238000004879 turbidimetry Methods 0.000 description 1
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- 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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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/22—Klebsiella
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Abstract
The invention relates to a method for producing 1,3-propylene glycol by fermentation based on cofactor regulation, 1,3-propylene glycol is produced by fermenting klebsiella pneumoniae, and riboflavin is added into a culture medium for fermentation culture; or the Klebsiella pneumoniae and the Shewanella are mixed and fermented to produce 1,3-propylene glycol. In addition, the invention discovers that the mixed fermentation of the Klebsiella pneumoniae and the Shewanella can realize mixed fermentation, the mixed fermentation of the Klebsiella pneumoniae and the Shewanella and the improvement effect of the single addition of the riboflavin on the yield of 1,3-propylene glycol are close to each other, but the yield of the byproducts is reduced to a greater extent, and huge economic benefits are brought to the industrial production of 1,3-propylene glycol.
Description
Technical Field
The invention belongs to the technical field of microbial fermentation, and particularly relates to a method for producing 1,3-propylene glycol through fermentation regulation and control based on a cofactor.
Background
1,3-propanediol (1,3-PDO) is a versatile chemical feedstock that can be used to synthesize polyesters, polyurethanes, polyethers, and heterocyclic compounds, and is widely used in the fields of food, pharmaceuticals, cosmetics, and biodegradable plastics. For example, 1,3-propanediol can be used as a monomer to synthesize a novel polyester material, polytrimethylene terephthalate (PTT), which has a wide market prospect due to its excellent performance and wide application.
5363 the chemical synthesis of 1,3-propanediol has two main methods: one is the hydroformylation of ethylene oxide to 3-hydroxypropanal (3-HPA) and the catalytic hydrogenation to 1,3-propanediol. In another, acrolein is converted to 3-hydroxypropanal by hydration and then further converted to 1,3-propanediol by catalytic hydrogenation. These chemical catalytic processes require expensive catalysts and can produce toxic intermediates. In contrast to chemical synthesis methods, biofermentation methods can employ renewable resources (glycerol, glucose, etc.) to produce 1,3-propanediol, typically under mild and environmentally friendly conditions. Wherein, the glycerol is taken as a byproduct in the production process of the biodiesel, and the yield of the glycerol is greatly improved along with the continuous development of the biodiesel market. The high value of 1,3-propanediol and the plethora of glycerol markets have made the synthesis of 1,3-propanediol by biological methods using glycerol a research hotspot. To date, a series of microorganisms have been reported to produce 1,3-PDO from glycerol, glucose and other raw materials, including klebsiella, clostridium, citrobacter, enterobacter, etc. Among them, klebsiella has attracted much attention due to its clear genetic background and excellent glycerol utilization ability, showing the potential for large-scale production of 1,3-PDO. However, the biological method for producing 1,3-PDO has the problems of insufficient reducing power and many byproducts. From the glycerol reductive metabolic pathway, 1,3-PDO synthesized from glycerol requires two NADH molecules per molecule. Thus, the classical anaerobic fermentation process for the production of 1,3-PDO from glycerol is limited by the NADH content. In Klebsiella pneumoniae, NADH required for the synthesis of 1,3-PDO by the reductive metabolism of glycerol is mainly regenerated during the oxidation of glycerol to pyruvate. Pyruvate is further converted into 2,3-butanediol (2,3-BDO), lactic acid, ethanol, acetic acid and other byproducts in the cells. This process will competitively consume glycerol and NADH, thereby affecting the titer and yield of 1,3-PDO. While the formation of byproducts not only results in carbon loss, but also is toxic to the microbial cells and inhibits their growth.
Disclosure of Invention
The invention aims to provide a method for producing 1,3-propylene glycol by fermentation based on cofactor regulation.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for producing 1,3-propylene glycol by fermentation based on cofactor regulation and control comprises producing 1,3-propylene glycol by fermenting Klebsiella pneumoniae, and adding riboflavin into the culture medium of fermentation culture; or the Klebsiella pneumoniae and the Shewanella are mixed and fermented to produce 1,3-propylene glycol.
In a preferred embodiment, the riboflavin is added at a concentration of 0.5-1.5mM; preferably 1mM.
As a preferred embodiment, the temperature of the fermentation culture is 25-37 ℃; preferably 30 deg.c.
As a preferred embodiment, the medium formulation of the fermentation culture is: carbon source, K 2 HPO 4 ·3H 2 O 4.45 g/L，KH 2 PO 4 1.3 g/L，(NH 4 ) 2 SO 4 2 g/L，MgSO 4 ·7H 2 O 1.6 g/L，CaCO 3 4 g/L and a microelement solution of 1 mL/L;
the microelement solution comprises FeSO 4 ·7H 2 O 5 g/L，CaCl 2 2 g/L，ZnCl 2 0.14 g/L，MnCl 2 ·4H 2 O 0.2 g/L，H 3 BO 3 0.12 g/L，CoCl 2 ·6H 2 O 0.4 g/L，CuCl 2 ·2H 2 O 0.04 g/L，NiCl 2 ·6H 2 O 0.05 g/L， Na 2 MoO 4 ·2H 2 O 0.07 g/L。
As a preferred embodiment, the carbon source in the culture medium of the fermentation culture is glycerol, and the concentration is 40-80 g/L; preferably 50-60 g/L.
As a preferred embodiment, the fermentation method is fed-batch fermentation, and when the carbon source concentration is reduced to 10g/L, the carbon source is added to 30 g/L.
As a preferred embodiment, the mode for producing 1,3-propylene glycol by using the fermentation of the Klebsiella pneumoniae and the Shewanella pneumoniae is as follows: after Klebsiella pneumoniae and Shewanella pneumoniae are respectively cultured by seeds, the seed liquid is inoculated to a fermentation medium for mixed fermentation.
In a preferred embodiment, the seed solution of Klebsiella pneumoniae and Shewanella pneumoniae is inoculated into the fermentation medium in equal proportion.
In a preferred embodiment, the culture medium for Shewanella seed culture is LB liquid medium.
As a preferred embodiment, the fermentation culture is carried out under anaerobic conditions.
In addition, the invention discovers that the mixed fermentation of the Klebsiella pneumoniae and the Shewanella can be realized, the Shewanella can produce riboflavin, the mixed fermentation of the Klebsiella pneumoniae and the Shewanella and the improvement effect of the single addition of the riboflavin on the yield of 1,3-propanediol are close to that of the mixed fermentation of the Klebsiella pneumoniae and the Shewanella on the yield of 1,3-propanediol, but the yield of the byproducts is reduced to a greater extent, and great economic benefits are brought to the industrial production of 1,3-propanediol.
Detailed Description
The Klebsiella pneumoniae used in the examples was Klebsiella pneumoniae (K.pneumoniae: (B))Klebsiella pneumoniae) KG1, purchased from China Industrial microbial cultures Collection management center with the collection number CICC 10781; shewanella is Shewanella (Shewanella)Shewanella oneidensis) MR-1, purchased from the American type culture Collection with deposit number ATCC 700550.
The biomass measuring method adopted by the invention comprises the following steps:
the biomass was measured by turbidimetry. Taking appropriate amount of culture brothReleasing a certain multiple, measuring the absorbance (absorbance value range is 0.2-0.8) at 600 nm by using a spectrophotometer, and multiplying the absorbance reading by the dilution multiple to obtain the OD of the culture solution 600 The value is obtained.
The determination method of the glycerol, 1,3-propylene glycol and the byproduct product adopted by the invention comprises the following steps:
the concentration of glycerol, 1,3-propanediol and byproduct product were determined using High Performance liquid chromatography (High Performance)
Liquid Chromatography, HPLC). The liquid chromatograph is a Saimer Fei Ultimate 3000, the detector is Shodex RI-101, the chromatographic column is a Burley AminexR HPX-87H organic acid and alcohol analysis column in the United states, the column temperature is 55 ℃, the mobile phase is 5mM sulfuric acid, the flow rate is 0.6 mL/min, and the sample feeding amount is 20 mu L.
Drawing a standard curve: analytically pure samples (glycerol, 1,3-propanediol, 2,3-butanediol, lactic acid, acetic acid, succinic acid and ethanol) were prepared as solutions with concentrations of 0.05 g/L, 0.1 g/L, 0.2 g/L, 0.4 g/L, 0.6 g/L, 0.8 g/L and 1 g/L, respectively, and were chromatographed together with the samples at each detection to draw a concentration-peak area standard curve.
Sample treatment: centrifuging at 12000 rpm for 6 min to obtain supernatant, diluting with mobile phase by corresponding times, filtering with 13 mm and 0.2 μm sterile needle filter to remove impurities, and measuring the contents of substrate and product in the fermentation liquid by liquid chromatography.
The examples relate to the following medium formulations:
klebsiella pneumoniae seed culture medium: glycerol 20 g/L, K 2 HPO 4 ·3H 2 O 4.45 g/L，KH 2 PO 4 1.3 g/L，(NH 4 ) 2 SO 4 2 g/L，MgSO 4 ·7H 2 O0.4 g/L, yeast powder 1 g/L, caCO 3 2 g/L, and microelement solution 1 mL/L (FeSO) 4 ·7H 2 O 5 g/L，CaCl 2 2 g/L，ZnCl 2 0.14 g/L，MnCl 2 ·4H 2 O 0.2 g/L，H 3 BO 3 0.12 g/L，CoCl 2 ·6H 2 O 0.4 g/L，CuCl 2 ·2H 2 O 0.04 g/L，NiCl 2 ·6H 2 O 0.05 g/L， Na 2 MoO 4 ·2H 2 O 0.07 g/L）。
Fermentation medium: glycerol 40-80g/L, K 2 HPO 4 ·3H 2 O 4.45 g/L，KH 2 PO 4 1.3 g/L，(NH 4 ) 2 SO 4 2 g/L，MgSO 4 ·7H 2 O 1.6 g/L，CaCO 3 4 g/L, microelement solution 1 mL/L. The trace element solution has the same components as those in the seed culture medium.
Shewanella seed culture medium: 5 g/L yeast powder, 10g/L peptone and 10g/L NaCl.
In the embodiment, different batches of fermentation exist, so that the fermentation results are not completely the same although the fermentation conditions are the same in a part of fermentation experiments, and the difference of the fermentation results among different batches is within +/-1%.
The following examples illustrate the invention in detail.
Example 1
The Klebsiella pneumoniae KG1 was cultured by fermentation using a fermentation medium, and the glycerol concentrations were 40 g/L, 50g/L, 60 g/L and 80g/L, respectively, in 4 experiments. 50 mL fermentation medium was prepared in 100 mL anaerobic bottles.
Preparing a seed solution: klebsiella pneumoniae seed medium was prepared in advance in test tubes and 250 mL Erlenmeyer flasks. The strain preserved at-80 deg.C is inoculated into a test tube containing seed culture medium at a ratio of 1%, and cultured in a shaker at a rotation speed of 180 rpm and a temperature of 37 deg.C to form 12 h. Then, the obtained culture was inoculated at a ratio of 1% into an Erlenmeyer flask containing a seed medium, and 8 h was cultured under the same conditions so that the cells reached the middle and late stages of the logarithmic phase as a fermentation seed solution.
Inoculating the seed liquid into a fermentation medium at an inoculation ratio of 10% (V/V), and performing shake cultivation at a rotation speed of 180 rpm and a temperature of 37 ℃ for 72 h.
TABLE 1 fermentation results of example 1
| Glycerol concentration (g/L) | 1,3 propylene glycol (g/L) | 2,3 butanediol (g/L) | Ethanol (g/L) | Lactic acid (g/L) | OD 600 |
| 40 | 20.29 | 7.97 | 5.39 | 3.28 | 2.46 |
| 50 | 22.68 | 8.68 | 4.69359 | 5.07537 | 2.56 |
| 60 | 21.34 | 8.45 | 3.76201 | 5.28256 | 2.53 |
| 80 | 20.21 | 8.36 | 3.9822 | 5.9251 | 2.34 |
Example 2
The effect of different fermentation temperatures on yield was investigated in example 2. The glycerol concentration was 50g/L, the fermentation temperatures were 25 ℃, 30 ℃, 37 ℃ and 40 ℃ respectively, and other conditions were the same as in example 1.
Table 2 fermentation results of example 2
| Fermentation temperature (. Degree.C.) | 1,3-propanediol (g/L) | 2,3 butanediol (g/L) | Ethanol (g/L) | Lactic acid (g/L) | OD 600 |
| 25 | 24.23 | 8.78 | 5.50 | 2.03 | 2.81 |
| 30 | 26.63 | 8.71 | 4.85 | 3.04 | 2.79 |
| 37 | 22.70 | 7.20 | 3.97 | 5.35 | 2.48 |
| 40 | 16.12 | 6.80 | 3.76 | 7.12 | 2.29 |
Example 3
In example 3, the effect of different amounts of riboflavin added on the yield was investigated. In 5 experiments, the fermentation medium was supplemented with riboflavin at 0, 0.5, 1, 1.5, mg/L, respectively, and the other conditions were the same as in example 1.
TABLE 3 fermentation results of example 3
| Riboflavin (mM) | 1,3-propanediol (g/L) | 2,3 butanediol (g/L) | Ethanol (g/L) | Lactic acid (g/L) | Yield (g/g) |
| 0 | 24.93 | 8.29 | 4.35 | 3.931 | 0.33 |
| 0.5 | 25.92 | 8.33 | 4.15 | 4.41 | 0.38 |
| 1 | 28.01 | 8.38 | 3.40 | 4.70 | 0.41 |
| 1.5 | 26.56 | 8.15 | 3.80 | 4.33 | 0.37 |
Example 4
Fed-batch fermentation was carried out in a 5L fermentor, with an inoculum size of 10% (V/V), an initial liquid charge of 2.5L, a stirrer speed of 250 rpm, a fermentation temperature of 30 ℃ and a pH of 7.0 maintained by adjusting with 4 mol/L sodium hydroxide solution. The fermentation tank is not aerated, and anaerobic conditions are maintained. The glycerol concentration in the fermentation medium was 50 g/L. Control fermentation groups with and without added riboflavin were set up with 1mM riboflavin. When the concentration of the glycerol in the fermentation liquor is lower than 10g/L, the glycerol is added to 30 g/L for fermentation for 96h.
Table 4 fermentation results of example 4
| 1,3-propanediol | 2,3 butanediol | Ethanol | Lactic acid | Acetic Acid (AA) | Succinic acid | |
| Riboflavin-free group (g/L) | 60.76 | 13.54 | 2.39 | 9.69 | 5.63 | 7.82 |
| Adding riboflavin group (g/L) | 63.73 | 12.49 | 3.46 | 8.38 | 6.22 | 5.97 |
| Growth rate (%) | 4.89 | -7.76 | 44.77 | -13.43 | -10.48 | -23.66 |
Example 5
Preparing Shewanella seed liquid: seed media was prepared in advance in test tubes and 250 mL erlenmeyer flasks. The strain preserved at-80 deg.C is inoculated into a test tube containing seed culture medium in a proportion of 1%, and cultured in a shaker at the rotation speed of 180 rpm and the temperature of 37 deg.C to obtain 18 h. Then, the obtained culture was inoculated into an Erlenmeyer flask containing a seed culture medium at a ratio of 1%, 18-20 h was cultured under the same conditions, and the cells were allowed to reach the middle and late stages of the logarithmic phase as a fermentation seed solution.
The mixed system fed-batch fermentation with Shewanella added is carried out in a 5L fermentation tank, the inoculation amount of Klebsiella pneumoniae and Shewanella is 10% (V/V), the initial liquid loading amount is 2.5L, the stirring rotation speed is 250 rpm, the fermentation temperature is 30 ℃, and the pH is adjusted by 4 mol/L sodium hydroxide solution to maintain the pH at 7.0. The fermentation tank is not aerated, and anaerobic conditions are maintained. The glycerol concentration in the fermentation medium was 50 g/L. Meanwhile, a control fermentation group without adding Shewanella is arranged. When the concentration of glycerol in the fermentation broth is lower than 10g/L, glycerol is added to 30 g/L for fermentation 144h, and the fermentation results are shown in Table 5.
TABLE 5 fermentation results of example 5
| 1,3-propanediol | 2,3 butanediol | Ethanol | Lactic acid | Acetic Acid (AA) | Succinic acid | |
| Single-bacterium fermentation (g/L) | 59.82 | 13.76 | 2.47 | 9.71 | 5.68 | 7.81 |
| Mixed bacteria fermentation (g/L) | 62.89 | 10.25 | 3.05 | 6.86 | 5.23 | 6.49 |
| Growth rate (%) | 5.13 | -25.51 | 23.48 | -29.35 | -7.92 | -16.90 |
As can be seen from Table 5, 1,3-propanediol of 62.89 g/L can be produced by mixed fermentation, and the yield is improved by 5.13% compared with the fermentation result of single-strain fermentation. Meanwhile, the accumulation of the byproducts 2,3-butanediol, lactic acid, acetic acid and succinic acid is reduced. Besides, the yield of 1,3-propylene glycol is improved, compared with the case of adding riboflavin by mixed fermentation, the generation of byproducts is reduced to a greater extent, and the scheme can bring considerable economic benefits for the industrial application of producing 1,3-propylene glycol by using Klebsiella pneumoniae fermentation.
Compared with the fermentation result in the prior art, the fermentation process for adjusting the cofactor of the klebsiella pneumoniae improves the yield and the yield of 1,3-propylene glycol produced by the klebsiella pneumoniae and reduces the generation of byproducts.
Claims (10)
1. A method for producing 1,3-propylene glycol by fermentation based on cofactor regulation is characterized in that 1,3-propylene glycol is produced by fermenting Klebsiella pneumoniae, and riboflavin is added into a culture medium of fermentation culture; or the Klebsiella pneumoniae and the Shewanella are mixed and fermented to produce 1,3-propylene glycol.
2. The method according to claim 1, wherein the riboflavin is added at a concentration of 0.5-1.5mM.
3. The method of claim 1, wherein the temperature of the fermentation culture is 25-37 ℃.
4. The method of claim 1, wherein the fermentation medium is formulated as: carbon source, K 2 HPO 4 ·3H 2 O 4.45 g/L，KH 2 PO 4 1.3 g/L，(NH 4 ) 2 SO 4 2 g/L，MgSO 4 ·7H 2 O 1.6 g/L，CaCO 3 4 g/L and a microelement solution of 1 mL/L;
the microelement solution comprises FeSO 4 ·7H 2 O 5 g/L，CaCl 2 2 g/L，ZnCl 2 0.14 g/L，MnCl 2 ·4H 2 O 0.2 g/L，H 3 BO 3 0.12 g/L，CoCl 2 ·6H 2 O 0.4 g/L，CuCl 2 ·2H 2 O 0.04 g/L，NiCl 2 ·6H 2 O 0.05 g/L， Na 2 MoO 4 ·2H 2 O 0.07 g/L。
5. The method of claim 1 or 4, wherein the carbon source in the medium of the fermentation culture is glycerol at a concentration of 40-80 g/L.
6. The method of claim 5, wherein the fermentation process is fed-batch fermentation, and the carbon source is added to 30 g/L when the carbon source concentration is reduced to 10 g/L.
7. The method of claim 1, wherein the 1,3-propanediol is produced by the mixed fermentation of klebsiella pneumoniae and shewanella pneumoniae by the following method: after the Klebsiella pneumoniae and the Shewanella pneumoniae are respectively subjected to seed culture, the seed solution is inoculated to a fermentation medium for mixed fermentation.
8. The method according to claim 7, wherein the seed solution of Klebsiella pneumoniae and Shewanella pneumoniae is inoculated to the fermentation medium in equal proportion.
9. The method according to claim 7, wherein the culture medium for Shewanella seed culture is LB liquid medium.
10. The method of claim 1, wherein the fermentation culture is performed under anaerobic conditions.
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