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Screening method and application of strain for producing 1,3-propanediol

A screening method and strain technology, applied in the field of 1,3-propanediol, can solve problems such as hidden dangers of biological safety, and achieve the effects of reducing production costs, simple operation, and energy consumption

Active Publication Date: 2013-03-13
XIAMEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] 2. Chinese patent 200710048122.8 discloses a method for preparing 1,3-propanediol by fermenting glycerol with microorganisms. The strain used in this method is Klebsiella, which is an opportunistic pathogen and has hidden dangers in biological safety

Method used

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  • Screening method and application of strain for producing 1,3-propanediol
  • Screening method and application of strain for producing 1,3-propanediol

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] 1. Isolation and purification of strains:

[0037] 1) Take 1g of each collected sample, add it to 9mL cabbage juice, and culture for 24 hours at 37°C under sealed enrichment.

[0038] 2) Place the above-mentioned culture solution in a water bath at 80°C for 10 minutes to kill non-spore bacteria, transfer it to the Clostridium proliferation liquid medium, and culture it anaerobically at 37°C for 24 hours.

[0039] 3) Put the above-mentioned culture solution in a water bath at 80°C for 10 minutes, transfer it to the fermentation medium, and cultivate it for anaerobic selective enrichment at 37°C for 24 hours. The fermentation products were analyzed by high performance liquid chromatography, and the positive bacteria capable of producing 1,3-propanediol were selected for the next experiment.

[0040] 4) Gradually dilute the culture solution, adopt the high-layer agar column method (add 2% agar to the RCM medium, cover 2-3cm with paraffin oil), culture at 37°C for 24 hours...

Embodiment 2

[0057] (1) Fermentation method: 5L mechanical stirring fermenter, batch fermentation with nitrogen

[0058] (2) Strains: Clostridium butyricum Gen0

[0059] (3) Medium:

[0060] Seed medium ( / L) is: glycerol, 20g; K 2 HPO 4 ·3H 2 O, 4.45g; KH 2 PO 4 , 1.3g; (NH 4 ) 2 SO 4 , 2g; MgSO 4 ·7H 2 O, 0.2g; CaCl 2 2H 2 O, 0.02g; yeast powder, 1g; trace element solution, 1mL; Fe solution, 1mL; natural pH.

[0061] The fermentation medium ( / L) is: waste glycerol (glycerol content is about 90% w / w); KH 2 PO 4 , 1.2g; (NH 4 ) 2 SO 4 , 2g; MgSO 4 ·7H 2 O, 0.2g; CaCl 2 2H 2 O, 0.02g; yeast powder, 1g; trace element solution, 1mL; Fe solution, 1mL; pH 7.0.

[0062] The Fe solution ( / L) is: FeSO 4 ·7H 2 O, 5 g; HCl (37%), 4 mL.

[0063] The trace element solution ( / L) is: ZnCl 2 , 0.07g; MnCl 2 4H 2 O, 0.1g; H 3 BO 3 , 0.06g; CoCl 2 ·6H 2 O, 0.2g; CuCl 2 2H 2 O, 0.02g; NiCl 2 ·6H 2 O, 0.025g; Na 2 MoO 4 2H 2 O, 0.035 g; HCl (37%), 0.9 mL.

[0064] (4) F...

Embodiment 3

[0071] (1) Fermentation method: 5L mechanical stirring fermenter, batch fermentation with nitrogen gas.

[0072] (2) Strains: Clostridium butyricum Gen100.

[0073] (3) Culture medium: the same as in Example 2.

[0074] (4) Fermentation process:

[0075] Seed culture: with embodiment 2.

[0076] Seed expansion cultivation: with embodiment 2.

[0077] 5L fermenter culture: Inoculate the expanded seed liquid with 10% inoculum in 2L fermentation medium containing 70g / L waste glycerol, control the fermentation temperature at 37°C, and use a pH concentration of 4mol / L NaOH solution The automatic adjustment is maintained at 7.0, the stirring speed is 150rpm, and nitrogen (0.1vvm) is passed into the fermenter during the fermentation process to maintain the anaerobic condition of the fermentation system. When the pH of the fermentation broth rises significantly (higher than 7.0), the fermentation is terminated, and the fermentation time is 20 hours.

[0078] (5) Fermentation resu...

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Abstract

The invention discloses a screening method and application of a strain for producing 1,3-propanediol, relating to 1,3-propanediol. A screening method and application of clostridium butyricum Gen160 and the strain screening method for producing 1,3-propanediol with waste glycerol are provided. The screening method comprises the steps as follows: adding collected samples to cabbage juice, putting the samples into a water bath after an enrichment culture to kill nonsporeforms, transferring the samples to a clostridium multiplication liquid medium, carrying out an anaerobic culture and then putting the samples into the water bath again, transferring the samples to a fermentation medium, carrying out a high performance liquid chromatograph to analyze fermentation products after the anaerobic selective enrichment culture and selecting positive strains capable of producing 1,3-propanediol; separating out single colonies by a high layer agar column method; carrying out an aerobiotic culture and the anaerobic culture to remove facultative anaerobes and to obtain strict anaerobes, analyzing the fermentation products and selecting the strains to carry out 16S rDNA (ribosomal deoxyribonucleic acid) sequence analysis and identification, wherein the strains are capable of producing 1,3-propanediol and the cultural characteristics and the colonial morphologies confirm to the cultural characteristics of the clostridium butyricum consistently.

Description

technical field [0001] The invention relates to 1,3-propanediol, in particular to a screening method and application of strains producing 1,3-propanediol. Background technique [0002] 1,3-Propanediol is an important chemical raw material, which can be directly used as a synthetic raw material for antifreeze, various plasticizers, detergents, preservatives and emulsifiers, and can also be used in food, cosmetics and pharmaceutical industries. Its main use is as a monomer for synthesizing polymer materials such as polyester, polyether and polyurethane. At present, there are two main methods for the synthesis of 1,3-propanediol, namely chemical method and biological method. Among them, the production of 1,3-propanediol by chemical method has become increasingly mature, but the chemical method consumes non-renewable limited resources and causes environmental pollution. In recent years, the biotransformation method has been paid more and more attention by people because of its...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12N1/02C12P7/18C12R1/145
Inventor 方柏山陈斌朱春杰王世珍
Owner XIAMEN UNIV
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