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Screening method of organic nitrogen source for culture medium and for promoting growth of butyric acid bacillus methylotrophicus and methanol consumption

A technology of butyric acid bacteria and screening method, which is applied in the field of screening of organic nitrogen source for culture medium, and can solve the problems of low cell growth and low production capacity, etc.

Inactive Publication Date: 2021-07-16
NANJING UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Based on the problem of low cell growth caused by low production capacity in the methanol metabolism pathway, the effects of different nitrogen sources on cell growth and methanol utilization were investigated; the differences and changes in the proportion of amino acids during fermentation of different nitrogen source media were compared and analyzed to reveal the impact of amino acids on The key role of cell growth; the effect of key amino acids on cell growth is further confirmed by amino acid addition experiments. The invention provides a screening method for organic nitrogen sources used in culture media that promote the growth of butyricum methylovora and methanol consumption

Method used

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  • Screening method of organic nitrogen source for culture medium and for promoting growth of butyric acid bacillus methylotrophicus and methanol consumption
  • Screening method of organic nitrogen source for culture medium and for promoting growth of butyric acid bacillus methylotrophicus and methanol consumption
  • Screening method of organic nitrogen source for culture medium and for promoting growth of butyric acid bacillus methylotrophicus and methanol consumption

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1: Studying the Effects of Different Nitrogen Sources on Cell Growth and Methanol Utilization

[0024] Pick a 2mL centrifuge tube containing YTF medium from a single colony of Methylbutyricum bacterium (purchased from the ATCC Culture Collection Center) on the plate, and culture it overnight in an anaerobic box at 37°C to make it OD 600 After growing to 1, transfer to 25 mL PB medium according to 2% inoculum volume. Collect the fermentation broth every 12-24 hours, detect methanol by high-performance liquid chromatography, resuspend with 2ml ultrapure water, and detect its OD 600 . Wherein the HPLC detection method is: Agilent HPLC, the chromatographic column is Biorad HPX-87H, and the mobile phase is 0.8 g / L H 2 SO 4 solution, the column temperature was 60°C, the injection volume was 20 μL, the flow rate was 0.5 mL / min, and the methanol detector was a differential detector.

[0025] As can be seen from the results, the screened organic nitrogen sources, yea...

Embodiment 2

[0028] Example 2: Difference analysis of corn steep liquor and yeast powder amino acid components

[0029] Under the same nitrogen content, the addition of corn steep liquor and yeast powder had obvious differences in the growth of the strain. Investigate the differences of amino acids in each nitrogen source medium, and use MSTFA to derivatize and detect by GC / MS.

[0030] The derivatization method is as follows: put 1 mL of fermentation broth into a 2 mL centrifuge tube, centrifuge at 4000 g for 5 min, absorb 800 μL of the supernatant and place it in another clean centrifuge tube; draw 100 μL of the above supernatant into a clean centrifuge tube, add (0.14mg / mL deuterium-labeled succinic acid), and centrifuged briefly; after the sample was quickly frozen in liquid nitrogen, it was placed in a vacuum freeze dryer to freeze-dry overnight; Vortex the salt / pyridine solution (20mg / mL) and incubate in a 40°C water bath for 80 min; add 80 μL of MSTFA (N-methyl-N-(trimethylsilane) ...

Embodiment 3

[0034] Example 3: Effects of key amino acids on cell growth and methanol utilization:

[0035] According to the experimental results of Example 2, the amino acids with large differences in the fermentation time periods of corn steep liquor and yeast powder and obvious changes in the fermented content include glycine, alanine, and lysine. In order to verify the effects of different amino acids on the growth of the strain, the three amino acids were added to the original PB-M yeast powder medium to make the final concentration 5mM, and the fermentation of Butyricum methylovora (purchased from ATCC Culture Collection Center) was carried out. Growth characterization. Every 24 hours or so of fermentation, after aspirating the supernatant, pass the detection of OD 600 Check for growth.

[0036] The PB-M medium used contained potassium dihydrogen phosphate 4 g / L, dipotassium hydrogen phosphate 6 g / L, ammonium chloride 1 g / L, magnesium chloride hexahydrate 0.1 g / L, calcium chloride ...

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Abstract

The invention discloses a screening method of an organic nitrogen source for a culture medium and for promoting growth of butyric acid bacillus methylotrophicus and methanol consumption. The method comprises the following steps: respectively adding different nitrogen sources to inspect the influence on cell growth and methanol utilization, and screening out corn steep liquor and yeast powder which can obviously increase the methanol utilization by strains; and analyzing the difference in amino acid components of the two nitrogen sources, wherein glycine, alanine, lysine, proline and phenylalanine are greatly different in the two culture media in the fermentation process, alanine, glycine, lysine, glutamic acid and serine have a more obvious in change trend in the fermentation process, and strain fermentation growth characterization shows that the cell biomass is respectively increased by 60%, 50.2% and 44.1% when the glycine, the alanine and the lysine are added compared with the cell biomass without the amino acid.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a screening method for an organic nitrogen source used in a medium for promoting the growth of butyricum methylotroph and methanol consumption. Background technique [0002] Methylbutyricum ( Butyribacterium methylotrophicum ), bacteriologically classified as Clostridium, is an obligate anaerobic Gram-positive bacillus. B. methylotrophicum Simultaneous utilization of methanol, CO, CO 2 and other carbon compounds for fermentation. When a reduced substrate is provided, such as CH 3 OH-CO 2 、CH 3 OH-HCOOH, etc., namely the synthesis of butyric acid. Therefore, as a natural methanol-utilizing strain, methylbutyricum bacterium is different from aerobic methylotrophic bacteria. The reducing power of the methanol metabolic pathway consumes less and is mainly used for the accumulation of metabolites. At the same time, the strain has tolerance to methanol It has the advant...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/02G01N30/02G01N30/06G01N30/34G01N30/36G01N30/72C12R1/01
CPCC12Q1/025G01N30/02G01N30/06G01N30/34G01N30/36G01N30/7206G01N2333/195
Inventor 王昕秦家伦陈可泉王雪麟马琛
Owner NANJING UNIV OF TECH
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