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Method for producing polyhydroxyalkanoates (PHA) through sterilization-free fermentation

A technology of polyhydroxyalkanoate and hydroxybutyrate, applied in microorganism-based methods, biochemical equipment and methods, fermentation and other directions, can solve the problems of high production cost of PHA, restrict the development and promotion of PHA, etc., and reduce energy consumption. consumption, reduce production costs, and reduce costs

Active Publication Date: 2019-03-22
BLUEPHA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although higher biomass and PHA output can be achieved with the above two bacterial strains, the production cost of PHA is still very high, which seriously restricts the development and promotion of PHA.

Method used

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  • Method for producing polyhydroxyalkanoates (PHA) through sterilization-free fermentation
  • Method for producing polyhydroxyalkanoates (PHA) through sterilization-free fermentation
  • Method for producing polyhydroxyalkanoates (PHA) through sterilization-free fermentation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1 Halomonas (halomonas TD01) produces PHB in a 7.5L fermenter

[0047] Strain culture

[0048] 1. Strain activation: put the glycerol tube of Halomonas TD01 [preservation number: CGMCC No.4353, authorized in the patent application (patent announcement number: CN102120973B)] stored in the refrigerator at -80°C on a 60LB solid plate Single clones were isolated by streaking on the medium, and cultured in a 37°C incubator for 18-24h.

[0049] 2. First-class seeds: Take a single colony from the activated halomonas TD01 plate and place it in a 100mL shaker flask filled with 20mL60LB, and culture it in a shaking table at a temperature of 37°C and a rotation speed of 200rpm for 12h.

[0050] 3. Second-level seeds: The first-level seed solution is inoculated into a 500mL shaker flask filled with 100mL 60LB according to the inoculum amount of 1%, and cultivated in a shaker at a temperature of 37°C and a rotation speed of 200rpm for 10-12h.

[0051] 60LB medium compositi...

Embodiment 2

[0073] Example 2 Halomonas (halomonas TD40) produces P3HB4HB in a 7.5L fermentation system

[0074] Halomonas TD40 is based on the wild bacteria halomonas TD01 by synthetic biology technology, and introduces the 4-hydroxybutyryl-CoA transferase gene into the genome of TD01, so that it has the ability to synthesize P3HB4HB.

[0075] Strain culture

[0076] 1. Strain activation: Halomonas TD40 stored in a -80°C refrigerator (this strain has been disclosed in the following non-patent literature: Chen, X., Yin, J., Ye, J., Zhang, H., Che ,X.,Ma,Y.,Li,M.,Wu,L-P.,Chen,G-Q.,Engineering Halomonas bluephagenesis TD01for Non-sterile Production of Poly(3-hydroxybutyrate-co-4-hydroxybutyrate),Bioresource Technology(2017 ), and provided by Beijing Blue Crystal Microbiology Technology Co., Ltd.) Glycerol tubes were streaked on 60LB solid plate medium to isolate single clones, and cultured in a 37°C incubator for 18-24h.

[0077] 2. First-class seeds: Inoculate a single colony from the act...

Embodiment 3

[0104] Embodiment 3 halomonas (halomonas TD40) in 1m 3 Production of P3HB4HB in fermentors

[0105] Strain culture

[0106] 1. Strain activation: Streak the glycerol tubes of Halomonas TD40 stored in -80°C refrigerator on a 60LB solid plate medium to isolate single colonies, and culture them in a 37°C incubator for 18-24h.

[0107] 2. First-class seeds: Take a single colony from the activated Halomonas TD40 plate and place it in a 100 mL shaker flask filled with 20 mL60LB, and culture it in a shaker at a temperature of 37° C. and a rotation speed of 200 rpm for 12 hours.

[0108] 3. Second-level seeds: The first-level seed liquid is inoculated in a 2L shaker flask with 400mL 60LB according to the inoculum amount of 1%, and cultivated in a shaker at a temperature of 37°C and a rotation speed of 200rpm for 10-12h.

[0109] 4. Third-level seeds: The second-level seed liquid is inserted into a 100L fermenter according to the inoculum amount of 2%. Reduce the speed to 30% or les...

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Abstract

The invention provides a method for producing polyhydroxyalkanoates (PHA) through halophilic bacterium sterilization-free fermentation. The method comprises the following steps of (1) inoculating halophilic bacteria into a culture medium for fermentation culture; (2) after the fermentation to a logarithmic phase, supplementing a first supplementing material culture medium for first stage materialsupplementation fermentation; (3) after the first stage material supplementation fermentation is completed, supplementing a second supplementing material culture medium for second stage material supplementation fermentation; (4) after the second stage material supplementation fermentation is completed, supplementing a third supplementing material culture medium for third stage material supplementation fermentation until the fermentation is completed, wherein the first supplementing material culture medium in step (2) and the second supplementing material culture medium in step (3) comprise carbon sources and nitrogen sources; the third supplementing material culture medium in step (4) only comprises carbon sources; in addition, the nitrogen source content in the second supplementing material culture medium in step (3) is lower than the nitrogen source content in the first supplementing material culture medium in step (2).

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for producing polyhydroxyalkanoate (PHA) by non-sterile fermentation with halophilic bacteria. Background technique [0002] Since the last century, the modern industry developed on the basis of fossil raw materials such as coal and petroleum has brought unprecedented development and prosperity to mankind, but it has also caused great damage and pollution to the environment. Taking plastic as an example, plastic is an unprecedented new material invented by human beings on the basis of petrochemical industry, which brings great convenience to people's life. But then there are new problems. Our existing plastic products that use petroleum as raw material are completely non-degradable in nature, causing serious pollution to the soil. Therefore, people have been seeking to use degradable materials to replace traditional plastics, and polyhydroxyalkanoate is one of them. [000...

Claims

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Application Information

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IPC IPC(8): C12P7/62C12R1/01
CPCC12P7/625
Inventor 叶健文汪东升陈风义
Owner BLUEPHA CO LTD
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