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A method for producing 1,3-propanediol by fermentation of glycerol with mixed bacteria

A technology of mixed bacterial fermentation and propylene glycol, applied in the field of bioengineering, can solve the problems of not completely preventing production, inhibiting cell growth, increasing lactic acid, etc., and achieves the effects of simple operation, good substrate tolerance and high production intensity.

Active Publication Date: 2017-10-20
DALIAN UNIV OF TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Cui et al. also proved that knocking out the 2,3-butanediol pathway tends to increase the production of lactic acid, and excess lactate will prevent cells from using the excess NADH obtained by knocking out the 2,3-butanediol pathway, and will inhibit cell growth , thereby affecting the production of 1,3-propanediol (Journal of Applied Microbiology,2014,117:690-698)
Wu et al. inactivated acetoin reductase by inserting the formate dehydrogenase gene, thereby reducing 2,3-butanediol and formic acid, but did not completely prevent their production (Journal of Biotechnology, 2013, 168:194-200)

Method used

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  • A method for producing 1,3-propanediol by fermentation of glycerol with mixed bacteria
  • A method for producing 1,3-propanediol by fermentation of glycerol with mixed bacteria
  • A method for producing 1,3-propanediol by fermentation of glycerol with mixed bacteria

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Example 1 Mixed bacteria screening, strain isolation and identification

[0058] 1. Screening of mixed bacteria

[0059] Randomly collect 40 samples of barnacles, seaweed, sponges and sea mud in the sea area near Dalian, in a sterile ultra-clean bench, get 2g of each sample (barnacles, seaweeds, sponges need to be ground into a slurry with a sterile mortar, sea ​​mud without pretreatment), were added to 250mL Erlenmeyer flasks containing 50mL enriched medium, and the bottle mouths were sealed with gauze and kraft paper. Cultivate on a shaker at 37°C and 200r / min for 24 hours to obtain the primary culture; then inoculate 1 mL of the primary culture into another Erlenmeyer flask containing the seed medium, and cultivate under the same conditions for 24 hours to obtain the secondary culture; The third enrichment was carried out under the same conditions.

[0060] The enriched bacterial liquid of each sample obtained above was screened by shaking flask fermentation, and t...

Embodiment 2

[0066] The batch fermentation of embodiment 2 mixed bacteria NHN8

[0067] The mixed bacteria NHN8 obtained in Example 1 (the thalline number ratio of three kinds of bacteria contained therein is W3:Y1:Y5=208:17:82) is inserted in the 250mL Erlenmeyer flask that 100mL seed culture medium is housed, bottle The mouth was sealed with gauze and kraft paper, and the seeds were cultured in a shaker at 37°C and 200r / min for 12 hours to obtain the seed culture solution; then the seed culture solution was inoculated into a 5L fermenter with 2L fermentation medium at a volume ratio of 10% , the fermentation medium is steam sterilized in advance and then cooled. Fermentation was carried out at 37°C, 250r / min, nitrogen flux 0.2vvm, and pH 7.0. During the fermentation process, take the fermentation broth every 1 or 2 hours, measure the glycerol concentration with sodium periodate oxidation method, measure the OD of the bacteria with spectrophotometry, and centrifuge the fermentation broth...

Embodiment 3

[0069] The batch fermentation of embodiment 3 single bacterium W3

[0070] According to the method described in Example 2, the single bacterium W3 obtained in Example 1 was inserted into the seed medium and cultivated for 12 hours to obtain the seed culture solution, and then the seed culture solution was inoculated into a 2L fermentation tank according to the volume ratio of 10%. The medium was fermented in a 5L fermenter at 37°C, 250r / min, nitrogen flux 0.2vvm, and 5M NaOH to maintain the pH at 7.0. During the fermentation process, the fermentation broth was taken every 1 or 2 hours to measure the glycerol concentration and cell OD, and the fermentation broth was centrifuged at 10000r / min for 10 minutes, and the supernatant was taken for high-performance liquid chromatography analysis.

[0071] Fermentation results such as image 3 As shown, the glycerin of 40g / L was consumed by strain W3 in 5.3 hours of fermentation, the PDO concentration reached 17.84g / L, the production i...

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Abstract

The invention provides a method for producing 1,3-propanediol through mixed bacterium fermentation glycerinum. According to the method, Klebsiella pneumoniae W3, Klebsiella pneumoniae Y1 and Klebsiella pneumoniae Y5 with the preservation numbers being CGMCC NO.10440, CGMCC NO.10439 and CGMCC NO.10438 are in mixed fermentation in a fermentation medium with the glycerinum as a substrate to produce the 1,3-propanediol. Mixed bacteria are good in substrate tolerance, by-products of fermentation products are fewer, especially 2,3-butanediol is not generated, and the 1,3-propanediol as the main product can be separated. By the adoption of the method, the defect that a single bacterial strain is poor in substrate tolerance is overcome, production efficiency is improved, the products do not contain the 2,3-butanediol, the problem generated in following separation is avoided, the mixed bacteria can tolerate the concentration of the glycerinum of 200 g / L, and the yield of the 1,3-propanediol reaches 81.40 g / L. The method can hopefully provide a simple and economic fermentation process for industrialization of production of the 1,3-propanediol in a microbiological fermentation method.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, in particular to a method for producing 1,3-propanediol by fermentation of mixed bacteria. Background technique [0002] 1,3-Propanediol (PDO) is an important chemical raw material, widely used in ink, printing and dyeing, paint, lubricant, antifreeze and other industries, and can also be used as an intermediate in medicine and organic synthesis. 1,3-propanediol can also be used as a polymer monomer to synthesize polymer materials, especially the new polyester fiber polytrimethylene terephthalate (PTT) with excellent synthetic performance. PTT is considered to be a new type of polyester material that combines the high performance of polyethylene terephthalate (PET) and the ease of processing of polybutylene terephthalate (PBT), and has the potential of natural circulation. Biodegradable properties are currently a hot spot in the development of new varieties of synthetic fibers. These all...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P7/18C12R1/22
CPCC12P7/18
Inventor 修志龙刘会芳陈洋姜莉莉孙亚琴
Owner DALIAN UNIV OF TECH
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