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Ammonium ion resistant colibacillus for producing succinic acid and application thereof

A technology for producing succinic acid and Escherichia coli, which is applied in the direction of bacteria, microorganism-based methods, microorganisms, etc., can solve problems such as unsatisfactory fermentation effect, high price, and complicated extraction process, and achieve great social significance and economic value.

Active Publication Date: 2015-09-02
态创生物科技(广州)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the pH regulators required by the process that can be realized mainly include sodium salts, calcium salts, magnesium salts, and concentrated ammonia water. The first three have disadvantages such as large reagent consumption, high price, and complicated downstream extraction processes. In the succinic acid process, there is no need to consume a large amount of reagents, and the production of by-products is less. Crystallized succinic acid is the only product. This process has the advantages of closure, cleanness, cheapness and high efficiency.
[0006] This application uses the strain BER208 independently screened and preserved by our laboratory as the starting strain (preservation number CCTCC NO: M2012351). Since ammonium ions have an inhibitory effect on the growth and acid production of the strain, the effect of pH adjustment with ammonia water is not ideal.

Method used

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  • Ammonium ion resistant colibacillus for producing succinic acid and application thereof
  • Ammonium ion resistant colibacillus for producing succinic acid and application thereof
  • Ammonium ion resistant colibacillus for producing succinic acid and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] This example illustrates the method for continuous culture of Escherichia coli origin strain BER208.

[0035] The method for the continuous culture of Escherichia coli starting strains is as follows:

[0036] The starting strain of Escherichia coli BER208 (self-screened and preserved in this laboratory, preservation number CCTCCNO: M2012351) was activated in LB test tubes, and cultured overnight at 37°C, 200r / min; Inserted into shake flasks, cultured at 37°C, 200r / min for 6h, as secondary seeds. The secondary seed liquid is inoculated into the continuous culture device with an inoculum of 10%. When the cell density in the device rises and reaches a steady state, the dilution rate is increased, and according to the changes in the cell density and sugar concentration in the reactor, Constantly adjust the dilution rate. When the cell density and sugar consumption in the device remain unchanged for 3 retention times, it indicates that mutant strains may appear. Samples ar...

Embodiment 2

[0038] This example illustrates the method of screening excellent Escherichia coli.

[0039] Wherein, the medium formula used is as follows:

[0040] Solid synthetic medium plate: citric acid 3g / L, Na 2 HPO 4 12H 2 O4g / L, KH 2 PO 4 8g / L, MgSO 4 ·7H 2 O1g / L, CaCl 2 2H 2 O10.0mg / L, ZnSO 4 ·7H 2 O0.5mg / L, CuCl 2 2H 2 O0.25mg / L, MnSO 4 ·H 2 O2.5mg / L, CoCl 2 ·6H 2 O1.75mg / L, H 3 BO 3 0.12mg / L, Al 2 (SO4) 3 1.77mg / L, Na 2 MoO 4 2H 2 O0.5mg / L, ferric citrate 16.1mg / L, agar 20g / L, glucose 10g / L, with (NH 4 ) 2 HPO 4 Provide ammonium ions and make NH 4 + The concentration is 0.6mol / L, and the addition amount is 40g / L.

[0041] Seed medium: peptone 10g / L, yeast powder 5g / L, NaCl 5g / L.

[0042] Shake flask fermentation medium: citric acid 3g / L, Na 2 HPO 4 12H 2 O4g / L, KH 2 PO 4 8g / L, MgSO 4 ·7H 2 O1g / L, CaCl 2 2H 2 O10.0mg / L, ZnSO 4 ·7H 2 O0.5mg / L, CuCl 2 2H 2 O0.25mg / L, MnSO 4 ·H 2 O2.5mg / L, CoCl 2 ·6H 2 O1.75mg / L, H 3 BO 3 0.12mg / L, Al 2...

Embodiment 3

[0054] This example illustrates the passage stability of the mutant strain BER528

[0055] On the synthetic medium plate, the mutant strain BER528 was passaged 30 times, and the strain was added with 0.53mol / LNH in exogenous sources every 5 times 4 + Fermentation verification was carried out respectively in the shake flasks of the same experiment, and the experimental results are as shown in Table 2:

[0056]Table 2 Stability analysis of mutant strain BER528 passage

[0057]

[0058] From the experimental results, it can be seen that after 6 consecutive passages, the succinic acid production and DCW of the mutant strain BER528 are relatively stable, and it has good passage stability, which can be used as a production strain for further research and development.

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Abstract

The invention discloses an ammonium ion resistant colibacillus for producing succinic acid and application thereof and belongs to genus Escherichia. The colibacillus is named as Escherichia coli BER528, and the accession number CCTCC NO is of M2015160. On the anaerobic condition, the strain is capable of resisting high-density ammonium ions and accumulating succinic acid. During anaerobic shake flask fermentation with 0.53mol / LNH4 + added externally, 72h strain DCW is of 1.82g / L, the succinic acid yield is of 11.72g / L, but an original parent strain grows extremely slow, the 72h strain DCW is of 1.14g / L only, and the succinic acid yield is of 2.56g / L. Compared with that of the parent strain, the succinic acid yield is increased by 4.57 times. In the process of anaerobic fermentation with pH regulation through ammonia water by the aid of a 5L fermentation tank for 90h, the mutant strain DCW and final succinic acid yield are of 1.22g / L and 27.32g / L and is of 1.16 times and 1.88 times of those of the parent strain, and the mutant strain BER528 has important social benefit and economic value accordingly.

Description

technical field [0001] The invention relates to an ammonium ion-tolerant succinate-producing Escherichia coli and its application, and belongs to the technical field of industrial microorganism breeding and fermentation. Background technique [0002] Succinic acid, also known as succinic acid, is a common natural organic acid widely found in the human body, animals, plants and microorganisms. As an intermediate product of the TCA cycle and one of the terminal reduction products of anaerobic metabolism, succinic acid plays a very important role in the process of biological metabolism. Research results in recent years have shown that succinic acid can also be used as a C4 platform compound to synthesize bulk chemicals such as 1,4-butanediol, tetrahydrofuran, and γ-butyrolactone, as well as polybutylene succinate (PBS) biodegradable compounds. Degrades polyester. In recent years, with the increasing depletion of fossil resources and the increasingly serious environmental prob...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12P7/46C12R1/19
CPCC12P7/46C12R2001/19C12N1/205
Inventor 姜岷管钊马江锋吴明科陈吴方陈美丽
Owner 态创生物科技(广州)有限公司
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