Method for producing PHA by culturing halophilic bacteria with low-salt culture medium

A technology for halophilic bacteria and culture medium, applied in the field of culturing halophilic bacteria with low-salt culture medium to produce PHA, can solve the problems of increasing the production cost of PHA and the high processing cost

Active Publication Date: 2021-11-02
TSINGHUA UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Due to the high salt concentration of wastewater, the treatm

Method used

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  • Method for producing PHA by culturing halophilic bacteria with low-salt culture medium
  • Method for producing PHA by culturing halophilic bacteria with low-salt culture medium
  • Method for producing PHA by culturing halophilic bacteria with low-salt culture medium

Examples

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Effect test

Embodiment 1

[0087] Example 1: Low-salt medium culture produces halophilic bacteria cells containing PHB, and extracts and purifies PHA from the fermentation broth

[0088] In the present invention, by adding glucose as a carbon source in halophilic bacteria (see Table 1), the PHA synthase ( phaC ) under the action of its own metabolism to produce 3HB-CoA and polymerize into PHB.

[0089] Cultivate TD01, TD01AB, TDH4P, TDH4ABP, TD68, TD68AB, TD68-194 and TD68-194AB in 10LB respectively, and TD01, TDH4P, TD68 and TD68-194 in 60LB medium at 37°C and 200 rpm for 12 hours After that, they were inoculated into 50mL 10MMG and 60MMG medium at 1%, respectively, and 30g / L glucose was added as a carbon source at the same time, and cultured for 48 hours. After 48 hours, the bacteria were collected, and the dry cell weight and PHA content were detected. Three parallel samples were set for each experiment, and the results were averaged. The results are shown in Table 1 below:

[0090] Table 1 Vario...

Embodiment 2

[0093] Example 2: Cultivate and produce halophilic bacteria cells containing P3HB4HB in low-salt medium, and extract and purify PHA from the fermentation broth

[0094] The invention passes through the Halomonas bluephagenesis 4-Hydroxybutyryl-CoA transferase ( orf Z ) to synthesize 4HB-CoA, and in the cell's own PHA synthase ( phaC ) under the action of 3HB-CoA produced by its own metabolism to form P(3HB-co-4HB).

[0095] Put TD01, TD01AB, TDH4P, TDH4ABP, TD68, TD68AB, TD68-194 and TD68-194AB in 10LB respectively, and TD01, TDH4P, TD68 and TD68-194 respectively in 60LB medium at 37°C, 200rpm, after 12 hours of culture , were inoculated into 50mL 10MMG and 60MMG medium at 1%, respectively, and 5g / L γ-butyrolactone was added as a carbon source at the same time, and cultured for 48 hours. After 48 hours, the bacteria were collected, and the dry cell weight and PHA content were detected. Three parallel samples were set for each experiment, and the results were averaged. T...

Embodiment 3

[0099] Example 3: Increase the content of P3HB4HB and cell dry weight in the fermentation broth of halophilic bacteria in low-salt medium. (by Halomonas bluephagenesis TDH4P series as an example)

[0100] The invention passes through the Halomonas bluephagenesis 4-Hydroxybutyryl-CoA transferase ( orf Z ) to synthesize 4HB-CoA, and in the cell's own PHA synthase ( phaC ) under the action of 3HB-CoA produced by its own metabolism to form P(3HB-co-4HB). On this basis, ARTP mutagenesis and low-salt stress screening were carried out, and PHA synthase from Eutropha rosenbergii was overexpressed on the plasmid and genome respectively ( phaC ), β-ketothiolase ( phaA ), NADH-type acetoacetyl-CoA reductase ( phaB ), the ultimate success was in obtaining strains that could grow and accumulate large amounts of PHA in low-salt media. Both dry weight and PHA content were increased.

[0101] Inoculate TDH4P, TDH4ABP, TDH4ABP+phaCAB (plasmid) and TDH4ABP+phaCAB (chromosome) in 1...

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Abstract

The invention provides a halophilic bacterium with the preservation number of CGMCC No.22795 and the preservation date of June 28, 2021. The invention further provides a preparation method of the halophilic bacterium, a method for producing PHA by culturing the halophilic bacterium with a low-salt culture medium and an application of the halophilic bacterium. In the method, the used halophilic bacterium can be obtained by breeding means of mutagenesis screening and molecular modification, and a method for producing PHA containing multiple monomers by adding a non-related carbon source (a substrate, such as glucose, which is not directly related to a microbial PHA structure) is realized in the low-salt culture medium, and the proportion of each monomer is adjusted according to the type of the carbon source. According to the method, the problems that traditional halophilic bacteria culture and separation steps are complicated, downstream high-salt sewage is difficult to treat and the like can be solved, and PHA is produced through open fermentation.

Description

technical field [0001] The present invention relates to the technical field of biochemistry, in particular to a new halophilic bacteria (preservation number: CGMCCNo.22795, classified as: Halomonas bluephagenesis , the preservation date is June 28, 2021), and the production of PHB, P3HB4HB, P(3HB-co-4HB-co-3HV) by culturing halophilic bacteria in low-salt medium with non-related carbon sources (such as glucose, etc. , PHBHHx, products such as homopolymer P3HP or copolymer P(3HB-co-3HP) (or PHBHP) containing 3HP, and methods for adjusting the monomer components of various PHAs. Background technique [0002] Polyhydroxyalkanoates (PHA) is a degradable polyester that can be accumulated by a variety of bacteria. According to its monomer composition, it can be divided into short-chain and / or medium- and long-chain PHA, and there are also homopolymers and copolymers. things. PHA is an environmentally friendly material with broad application prospects as an environmentally friend...

Claims

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Application Information

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IPC IPC(8): C12N1/20C12N1/21C12N15/53C12N15/54C12N13/00C12P7/62C12R1/01
Inventor 陈国强张李湛叶健文黄悟哲吴赴清兰宇轩
Owner TSINGHUA UNIV
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