Clostridium butyricum strain with high yield of 1,3-propanediol and sequential inoculation fermentation process

A technology of inoculation fermentation and Clostridium butyricum, applied in the field of bioengineering, can solve the problems of repeated batch fed-feed fermentation without Clostridium butyricum, long lag period of strain growth, poor fermentation ability, etc., to reduce fermentation steps and fermentation equipment investment, simplify fermentation steps, and solve the effect of long time

Active Publication Date: 2019-11-12
DALIAN UNIV OF TECH
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  • Description
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Problems solved by technology

In the above-mentioned repeated batch fermentation, the bacterial strains at the end of fermentation are mainly used as seed liquid, and fresh medium is injected for repeated fermentation. The strains at the end of fermentation have a long growth lag period, slow metabolism and poor fermentation ability, which lead to repeated batch fermentation of cyclic inoculation. The reason why only 4 batches can be repeated
It will be more difficult to further obtain high-concentration 1,3-propanediol through repeated batch-type fed-feed fermentation, and there is no report on the realization of repeated batch-type fed-feed fermentation of Clostridium butyricum
Therefore, overcoming the above-mentioned problem of poor seed quality in repeated fermentations is the key to promoting the continuous production of high-concentration 1,3-propanediol by Clostridium butyricum

Method used

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  • Clostridium butyricum strain with high yield of 1,3-propanediol and sequential inoculation fermentation process
  • Clostridium butyricum strain with high yield of 1,3-propanediol and sequential inoculation fermentation process
  • Clostridium butyricum strain with high yield of 1,3-propanediol and sequential inoculation fermentation process

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Experimental program
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Effect test

Embodiment 1

[0054] Example 1 Screening and domestication of Clostridium butyricum DL07 producing 1,3-propanediol

[0055] The anaerobic activated sludge comes from the anaerobic tank 1 of Dalian Dongtai Sewage Treatment Plant. In the anaerobic box, take 1 mL of anaerobic activated sludge, add it to an anaerobic bottle filled with 9 mL of sterile deaerated water, shake and mix evenly, and place it in a water bath at 80 °C for 10 min to kill non-spore bacteria. Use a sterile syringe to take 100 μL of the suspension and spread it on a solid seed medium plate, culture it anaerobically at 37°C, select a single colony that grows normally under anaerobic conditions, and perform subsequent streaking on the plate to purify the single bacteria. After 7 times of streaking on the plate, a single bacterium DL07 was finally isolated, and the colony morphology was as follows: figure 1 shown. After the single bacterium DL07 was enriched with the liquid seed medium, the bacterium was domesticated with t...

Embodiment 2

[0056] Embodiment 2 takes crude glycerol as the continuous feeding batch fed-batch fermentation of substrate

[0057] Put the Clostridium butyricum DL07 stored in the glycerol bottle at low temperature into the anaerobic bottle containing the seed medium at a 4% inoculum, and after culturing for 12 hours, transfer it to a new seed medium with a 2% inoculum, and cultivate 12h. After the two rounds of activation are completed, it is inserted into a fermenter with a 10% inoculum size for fermentation. Nitrogen gas was introduced at a rate of 0.1vvm 1h before inoculation, and adjusted to 0.05vvm 1h after inoculation until the end of fermentation. The fermentation medium uses crude glycerin, the initial glycerin concentration is 40g / L, and the yeast powder is 4g / L. During the fermentation process, the crude glycerol is continuously added to maintain the glycerol concentration at 10-25g / L. The fermentation time is 34h. The results are as follows: Figure 4 shown. The final 1,3-pr...

Embodiment 3

[0058] Embodiment 3 takes crude glycerol as the fed-batch fed-batch fermentation of substrate

[0059] The activated seed culture solution is inserted into a fermenter with 10% inoculum size to carry out fermentation. The fermentation medium has an initial crude glycerol concentration of 40 g / L and yeast powder of 2 g / L. In the batch-fed fermentation, it is divided into three stages of feeding, the first stage: when the glycerol concentration is lower than 20g / L, feed crude glycerol until the glycerin concentration is 40g / L; the second stage: when the glycerol concentration is lower than When the concentration of glycerol is 15g / L, feed crude glycerin until the concentration of glycerin is 65g / L; the third stage: when the concentration of glycerin is lower than 20g / L, feed crude glycerol to 60g / L; the fermentation time is 30h, and the results are as follows: Figure 5 shown. The final 1,3-propanediol concentration was 84.86g / L, the mass conversion rate was 0.521g / g, and the p...

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Abstract

The invention belongs to the technical field of bioengineering, and particularly relates to a clostridium butyricum strain with high yield of 1,3-propanediol and a sequential inoculation fermentationprocess thereof. The strain is named as clostridium butyricum DL07 and a preservation number is CGMCCCNO. 17934. The strain of the present invention can produce 104.78 g/L and 94.23 g/L of 1,3-propanediol by fermentation with pure glycerin and crude glycerin respectively, and the currently reported natural microorganisms uses the pure glycerin and crude glycerin to ferment and produce the highestyield of 1,3-propanediol. The invention also provides a sequential inoculation fermentation process and an automatic fed-substrate fermentation control method, which saves seed cultivation time and realizes multi-stage continuous fermentation, and provides a new fermentation control mode with high degree of automation and excellent economy for the production of 1,3-propanediol by microbial fermentation, and the process has the good industrial application prospect.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a high-yield 1,3-propanediol Clostridium butyricum and a sequential inoculation fermentation method. Background technique [0002] 1,3-Propanediol (1,3-PDO) is an important chemical raw material widely used in many industries such as pharmaceutical, food, cosmetics and textile industries. At the same time, 1,3-propanediol can be used as a polymer monomer to synthesize high-performance polymer materials, especially synthetic biodegradable polytrimethylene terephthalate (PTT). PTT is a new type of polyester fiber , has good softness, bulkiness and stain resistance, plus its own inherent elasticity, and good characteristics such as being able to be dyed at room temperature. It has huge market potential in the carpet and textile industries, and thus has become the latest development in the world. As one of the new polymer materials, 1,3 propanediol has a huge mark...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12P7/18C12Q3/00C12R1/145
CPCC12P7/18C12Q3/00C12R2001/145C12N1/205
Inventor 修志龙王晓丽周瑾洁沈俊涛孙亚琴郑亚峰牟英
Owner DALIAN UNIV OF TECH
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