阅读说明：本技术 一种生产1,3-丙二醇的方法 (Method for producing 1, 3-propylene glycol ) 是由 诸葛斌 王洁茹 谢梦梦 梁川 陆信曜 宗红 于 2019-09-30 设计创作，主要内容包括：本发明公开了一种生产1,3-丙二醇的方法,属于发酵技术领域以及生物技术领域。本发明提供了一种可用于发酵生产1,3-丙二醇的培养基,此培养基在满足可生产1,3-丙二醇的菌株在生产1,3-丙二醇过程中对无机盐的需求的前提下,使用少量(NH<Sub>4</Sub>)<Sub>2</Sub>HPO<Sub>4</Sub>替代了传统的用于发酵生产1,3-丙二醇的培养基中所有的如KH<Sub>2</Sub>PO<Sub>4</Sub>、(NH<Sub>4</Sub>)<Sub>2</Sub>SO<Sub>4</Sub>和MgSO<Sub>4</Sub>等的其他硫酸盐和磷酸盐,这大大降低了1,3-丙二醇生产过程中对硫酸盐和磷酸盐的消耗量,进而降低了1,3-丙二醇的生产成本,同时,降低了1,3-丙二醇生产过程中下游脱盐的压力。(The invention discloses a method for producing 1, 3-propylene glycol, belonging to the technical field of fermentation and the technical field of biology. The invention provides a culture medium for producing 1,3-propanediol by fermentation, which uses a small amount of (NH4)2HPO4 to replace all other sulfates and phosphates such as KH2PO4, (NH4)2SO4, MgSO4 and the like in the traditional culture medium for producing 1,3-propanediol under the premise of meeting the requirement of strains capable of producing 1,3-propanediol on inorganic salts in the process of producing 1,3-propanediol, thereby greatly reducing the consumption of sulfates and phosphates in the process of producing 1,3-propanediol, further reducing the production cost of 1,3-propanediol and simultaneously reducing the pressure of downstream desalination in the process of producing 1, 3-propanediol.)

1. A culture medium is characterized in that the components of the culture medium comprise 6-8 g/L of yeast extract, 6-15 g/L of glucose, 35-45 g/L of glycerol, (NH4)2HPO 40.5-4.5 g/L, FeSO 4.7H 2O 0.003.003-0.008 g/L, VB 120.01-0.02 g/L and 0.08-0.12 mL/L of trace element solution; the trace element solution comprises Na2MoO4 & 2H2O 0.30.30-0.4 g/L, CoCl2 & 6H2O 1.8.8-2.2 g/L, NiCl2 & 6H2O 0.20.20-0.3 g/L, H3BO30.5-0.7 g/L, MnCl2 & 4H2O 0.8.8-1.2 g/L, CuCl20.18-0.22 g/L and ZnCl20.6-0.8 g/L.

2. The culture medium of claim 1, wherein the culture medium comprises yeast extract 6g/L, glucose 12g/L, glycerol 40g/L, (NH4)2 hpo41.5g/L, FeSO 4.7H 2O 0.005.005 g/L, vb120.015g/L, and trace element solution 0.1 mL/L; the components of the trace element solution comprise Na2MoO4 & 2H2O 0.35.35 g/L, CoCl2 & 6H2O 2g/L, NiCl2 & 6H2O 0.25.25 g/L, H3BO30.6 g/L, MnCl2 & 4H2O 1g/L, CuCl20.2g/L and ZnCl20.7g/L.

3. A culture medium according to claim 1 or 2, wherein the pH of the culture medium is 7.3 to 7.6.

4. A culture medium according to any one of claims 1 to 3, wherein the pH of the culture medium is 7.5.

5. A method for producing 1,3-propanediol, characterized in that, the method comprises the steps of inoculating a strain capable of producing 1,3-propanediol into the culture medium of any one of claims 1-4 for fermentation to obtain a fermentation liquor, and then extracting the fermentation liquor to obtain the 1, 3-propanediol.

6. the method for producing 1,3-propanediol according to claim 5, wherein the 1, 3-propanediol-producing strain is Klebsiella Pneumoniae (Klebsiella Pneumoniae), Klebsiella oxytoca (Klebsiella oxytoca), Citrobacter Citrobacter (Citrobacter fluendii), and/or Clostridium butyricum (Clostridium butyricum).

7. The method for producing 1,3-propanediol according to claim 5 or 6, wherein the 1, 3-propanediol-producing strain is Klebsiella Pneumoniae (Klebsiella Pneumoniae).

8. A process for the production of 1,3-propanediol according to any of claims 5 to 7 wherein the fermentation temperature is from 35 to 38 ℃.

9. a process for the production of 1,3-propanediol according to any of claims 5 to 8 wherein the fermentation is carried out at a speed of from 80 to 120 rpm.

10. Use of a culture medium according to any one of claims 1 to 4 or a method according to any one of claims 5 to 9 for the production of 1, 3-propanediol.

Technical Field

the invention relates to a method for producing 1, 3-propylene glycol, belonging to the technical field of fermentation and the technical field of biology.

Background

1,3-propanediol (1,3-propanediol, 1,3-PDO) is a colorless, odorless, salty-tasting, and hygroscopic viscous liquid. It is a raw material for producing unsaturated polyester, plasticizer, surfactant, emulsifier and demulsifier; in the polyurethane industry, it is commonly used as a raw material of polyester polyol, an initiator of polyether polyol, a polyurethane chain extender, and the like; in the organic chemical industry, the monomer and the intermediate are also important, and the most important application is to synthesize polytrimethylene terephthalate (PTT) as a polymer monomer. The potential market capacity for 1,3-propanediol is expected to reach 227 million tons by 2020.

Currently, 1,3-propanediol is produced industrially by inoculating Klebsiella Pneumoniae (Klebsiella Pneumoniae) which can produce 1,3-propanediol into a fermentation medium to perform fermentation, and then extracting 1,3-propanediol from the fermentation broth. However, since Klebsiella Pneumoniae (Klebsiella Pneumoniae) has a high demand for SO 42-and PO 43-plasmas during the growth process, the fermentation medium used to culture Klebsiella Pneumoniae (Klebsiella Pneumoniae) often contains a large amount of inorganic salts that provide SO 42-and PO 43-plasmas to Klebsiella Pneumoniae (Klebsiella Pneumoniae), which not only greatly increases the production cost of 1,3-propanediol, but also remains in the fermentation broth at the end of fermentation (about 16-20% of the inorganic salts remain), which greatly increases the pressure of downstream desalination during the production of 1, 3-propanediol.

Therefore, it is urgently needed to find a method for producing 1,3-propanediol with low consumption of inorganic salts so as to reduce the production cost of 1,3-propanediol and simultaneously reduce the pressure of downstream desalination in the production process of 1, 3-propanediol.

disclosure of Invention

[ problem ] to

The technical problem to be solved by the invention is to provide a method for producing 1, 3-propylene glycol with low consumption of sulfate and/or phosphate.

[ solution ]

in order to solve the technical problem, the invention provides a culture medium, and the components of the culture medium comprise 6-8 g/L of yeast extract, 6-15 g/L of glucose, 35-45 g/L of glycerol, (NH4)2HPO 40.5-4.5 g/L, FeSO 4.7H 2O 0.003.003-0.008 g/L, VB 120.01-0.02 g/L and 0.08-0.12 mL/L of trace element solution; the trace element solution comprises Na2MoO4 & 2H2O 0.30.30-0.4 g/L, CoCl2 & 6H2O 1.8.8-2.2 g/L, NiCl2 & 6H2O 0.20.20-0.3 g/L, H3BO30.5-0.7 g/L, MnCl2 & 4H2O 0.8.8-1.2 g/L, CuCl20.18-0.22 g/L and ZnCl20.6-0.8 g/L.

In one embodiment of the invention, the components of the culture medium comprise 6g/L of yeast extract, 12g/L of glucose, 40g/L of glycerol, (NH4)2HPO41.5 g/L, FeSO 4.7H 2O 0.005.005 g/L, VB120.015 g/L and 0.1mL/L of trace element solution; the trace element solution comprises Na2MoO4 & 2H2O 0.35.35 g/L, CoCl2 & 6H2O 2g/L, NiCl2 & 6H2O 0.25.25 g/L, H3BO30.6 g/L, MnCl2 & 4H2O 1g/L, CuCl20.2 g/L and ZnCl20.7 g/L.

In one embodiment of the present invention, the pH of the culture medium is 7.3 to 7.6.

in one embodiment of the invention, the pH of the medium is 7.5.

The invention also provides a method for producing 1,3-propanediol, which comprises the steps of inoculating the bacterial strain capable of producing 1,3-propanediol into the culture medium for fermentation to obtain fermentation liquor, and then extracting the fermentation liquor to obtain the 1, 3-propanediol.

In one embodiment of the present invention, the 1, 3-propanediol-producing strain is Klebsiella Pneumoniae (Klebsiella Pneumoniae), Klebsiella oxytoca (Klebsiella oxytoca), Citrobacter Citrobacter (Citrobacter francisii) and/or Clostridium butyricum (Clostridium butyricum).

In one embodiment of the present invention, the 1, 3-propanediol-producing strain is Klebsiella Pneumoniae (Klebsiella Pneumoniae).

In one embodiment of the invention, the temperature of the fermentation is 35-38 ℃.

In one embodiment of the invention, the temperature of the fermentation is 37 ℃.

In one embodiment of the invention, the rotation speed of the fermentation is 80-120 rpm.

In one embodiment of the invention, the rotation speed of the fermentation is 100 rpm.

The invention also provides the application of the culture medium or the method in the production of 1, 3-propanediol.

[ advantageous effects ]

(1) The invention provides a culture medium for producing 1,3-propanediol by fermentation, which uses a small amount of (NH4)2HPO4 to replace all other sulfates and phosphates such as KH2PO4, (NH4)2SO4, MgSO4 and the like in the traditional culture medium for producing 1,3-propanediol by fermentation under the premise of meeting the requirement of strains capable of producing 1,3-propanediol on inorganic salts in the process of producing 1,3-propanediol, thereby greatly reducing the consumption of sulfates and phosphates in the process of producing 1,3-propanediol, further reducing the production cost of 1,3-propanediol and simultaneously reducing the pressure of downstream desalination in the process of producing 1, 3-propanediol; the consumption of sulfate and phosphate in the 1,3-propanediol produced by fermentation by using the culture medium is reduced by 87 percent compared with the consumption of sulfate and phosphate in the 1,3-propanediol produced by fermentation by using the traditional culture medium.

(2) The invention provides a method for producing 1,3-propanediol, which greatly reduces the consumption of sulfate and phosphate in the production process of 1,3-propanediol by using a culture medium which does not contain any other sulfate and phosphate except 1.5g/L (NH4)2HPO4 and 0.005g/L FeSO 4.7H2O, thereby reducing the production cost of 1,3-propanediol and simultaneously reducing the pressure of downstream desalination in the production process of 1, 3-propanediol.

Drawings

FIG. 1: changes in metabolites of Klebsiella Pneumoniae (Klebsiella Pneumoniae) in the absence of phosphate.

FIG. 2: the metabolites of Klebsiella Pneumoniae (Klebsiella Pneumoniae) vary under different phosphates.

FIG. 3: the nucleotide sequence of the gene budA, budB, budC, phoA and phoB of Klebsiella Pneumoniae (Klebsiella Pneumoniae) has changed transcription level under the phosphate-free condition.

FIG. 4: changes in the metabolites of Klebsiella Pneumoniae (Klebsiella Pneumoniae) in the absence of sulfate.

FIG. 5: changes in metabolites of Klebsiella Pneumoniae (Klebsiella Pneumoniae) in the co-absence of phosphate and sulfate.

FIG. 6: the metabolites of Klebsiella Pneumoniae (Klebsiella Pneumoniae) vary at different concentrations of corn steep liquor.

FIG. 7: the metabolites of Klebsiella Pneumoniae (Klebsiella Pneumoniae) varied at different concentrations (NH4) of 2HPO 4.

Detailed Description

Klebsiella Pneumoniae (Klebsiella Pneumoniae) referred to in the following examples was purchased from North Nara organism, product number BNCC 107352; the corn steep liquor referred to in the following examples was purchased from Jiangyin enzyme preparation works.

The media involved in the following examples are as follows:

Slant culture medium: 5g/L of yeast extract powder, 10g/L of sodium chloride, 10g/L of peptone and 20g/L of agar powder.

Seed culture medium: 5g/L of yeast extract powder, 10g/L of sodium chloride and 10g/L of peptone.

Original fermentation medium: 6g/L of yeast extract, 12g/L of glucose, 40g/L, MgSO42 g/L of glycerol, (NH4)2SO42g/L, KH2PO47.5 g/L, FeSO 4.7H 2O 0.005.005 g/L, VB120.015 g/L and 0.1mL/L of trace element solution, wherein the pH is adjusted to 7.5 by 10mol/L of KOH; wherein, the components of the trace element solution comprise Na2MoO 4.2H2O 0.35.35 g/L, CoCl 2.6H2O 2g/L, NiCl 2.6H2O 0.25.25 g/L, H3BO30.6 g/L, MnCl 2.4H2O 1g/L, CuCl20.2 g/L and ZnCl20.7g/L (the culture medium is the most commonly used fermentation medium for producing 1,3-propanediol by fermentation in industry and laboratories).

The fermentation processes referred to in the following examples are as follows:

Inoculating Klebsiella Pneumoniae (Klebsiella Pneumoniae) in a test tube slant culture medium in a streak manner, and culturing at 37 ℃ for 12-14 h to form a single colony; selecting single colony to inoculate into seed culture medium, culturing at 37 deg.C and 100rpm for 11h to obtain first-stage seed solution; transferring the primary seed solution into a new seed culture medium according to the inoculation amount of 1% (v/v), and culturing at 37 ℃ and 100rpm for 8h to obtain a secondary seed solution; and (3) transferring the secondary seed liquid into a fermentation medium according to the inoculation amount of 4% (v/v), and performing shake culture at 37 ℃ and 100rpm for 48 hours to obtain a fermentation liquid.

The detection methods referred to in the following examples are as follows:

Determination of biomass: the fermentation broth was measured for OD600 by UV spectrophotometer, and the equation for Dry Cell Weight (DCW) to the measured OD600 was 0.36g/L to 1OD 600.

Determination of metabolite content: centrifuging the fermentation liquid for 15min at 10000g, treating 500 μ L of supernatant with 0.22 μm water system microporous membrane, and detecting the concentrations of 1,3-PDO, 2,3-BDO, acetic acid, glycerol, succinic acid and lactic acid in the fermentation liquid by HPLC; wherein the chromatographic column is Aminex HPX-87H (300mm multiplied by 7.8mm, 9 μm), the mobile phase is 5mmol/L H2SO4, the column temperature is 60 ℃, the flow rate is 0.6mL min < -1 >, the sample injection volume is 20 μ L, and the detector is a differential refraction detector.

Determination of the relative transcription level of the genes: qRT-PCR method:

(1) The qPCR tube was configured with the system as in table 1:

TABLE 1 qRT-PCR System

2×AceQ Universal SYBR qPCR Mster Mix	10μL
		Upstream primer (10. mu. mol/L)	0.4μL
Downstream primer (10. mu. mol/L)	0.4μL
		cDNA(100μg/L)	1μL
ddH2O	to20μL

(2) The qPCR reaction was performed under the conditions of table 2:

TABLE 2 reaction conditions for qRT-PCR

And (3) taking fermentation liquor obtained by fermenting the original fermentation medium as a control group, repeating all qRT-PCR reactions for 3 times, and calculating the relative transcription level by adopting a 2-delta-Ct calculation mode.