Zymomonas mobilis mutant strains tolerant to high-concentration acetic acid and applications thereof

A Zymomonas, high-concentration technology, applied in the field of microorganisms to achieve rapid and efficient production and good growth performance

Active Publication Date: 2018-04-10
BIOGAS SCI RES INST MIN OF AGRI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the high concentration of acetic acid is still a huge challenge for the ethanol fermentation process in the lignocellulosic fuel ethanol fermentation process of Zymomonas mobilis.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] This embodiment illustrates the method for obtaining Zymomonas mobilis AQ8-1 and AQ8-9 by mutagenesis screening ZM4 as the starting strain:

[0030] 1) Irradiation mutagenesis was carried out on Zymomonas mobilis cells by the normal temperature plasma mutagenesis breeding technology. First, the starting strain ZM4 was activated and cultured at 30°C for 16 hours, and then the overnight culture (10 7 to 10 8 cells), centrifuge at 4°C for 5 min at 3000 rpm, wash the bacteria with normal saline and resuspend in 1 mL of normal saline; take 10 μL of the resuspended cells and place them in an ARTP mutagenesis breeding instrument for 120 s;

[0031] 2) Resuspend the cells after irradiation mutagenesis for 16 hours, and then spread them on a solid medium containing 7 g / L acetic acid for culture;

[0032] 3) All the colonies were selected and screened on a medium containing 8 g / L acetic acid. After repeated screening three times, two stable mutant strains were finally obtained, ...

Embodiment 2

[0034] Method for producing ethanol by fermenting mutant strains

[0035] 1) The preparation of fermentation medium: 50g / L glucose, 2g / L potassium dihydrogen phosphate, 10g / L yeast extract and 7g / L acetic acid, the pH of the medium at this time=3.92 (this pH value is added 7g / L pH reached naturally by acetic acid)

[0036] 2) Inoculate the strains on the fermentation medium and carry out fermentation culture: inoculate the strains AQ8-1, AQ8-9 and the starting strain on the medium respectively, the inoculum amount is 10%, and ferment and cultivate for 40-48 hours.

[0037] 3) ethanol is separated, and the conversion rate is calculated.

[0038] After 48 hours of fermentation and cultivation, the two mutant strains can consume all the glucose, and the ethanol conversion rates of the strains AQ8-1 and AQ8-9 reached 94% and 98% of the theoretical conversion rate respectively, while the conversion rate of the starting strain was only 12% of theoretical conversion.

Embodiment 3

[0040] Method for producing ethanol by fermentation of mutant strain

[0041] 1) Preparation of fermentation medium: 50g / L glucose, 2g / L potassium dihydrogen phosphate, 10g / L yeast extract, 8g / L acetic acid, pH3.86 (this pH value is the pH naturally achieved by adding 8g / L acetic acid )

[0042] 2) Inoculate the strains on the fermentation medium and carry out fermentation culture: inoculate the strains AQ8-1, AQ8-9 and the starting strain on the medium respectively, the inoculum amount is 10%, and ferment and cultivate for 60 hours.

[0043] 3) ethanol is separated, and the conversion rate is calculated.

[0044] After 60 hours, the two mutant strains could consume all the glucose, and the ethanol conversion rates of the strains AQ8-1 and AQ8-9 both reached 96% of the theoretical conversion rate, and the starting strains could not grow normally at this time.

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Abstract

Zymomonas mobilis mutant strains tolerant to high-concentration acetic acid and applications thereof are disclosed. The taxonomic name of one of the strains is zymomonas mobilis AQ8-1, and the accession number of the zymomonas mobilis AQ8-1 is GDMCC60258. The taxonomic name of the other strain is zymomonas mobilis AQ8-9, and the accession number of the zymomonas mobilis AQ8-9 is GDMCC60259. Mutation screening includes 1) performing room-temperature plasma induced mutation; 2) performing renewing culture by utilizing a medium; and 3) repeatedly screening under a high acetic acid concentration to obtain the strains. The strains can rapidly grow in a culture liquid containing 5.0-8.0 g/L of acetic acid, and can perform alcoholic fermentation. The invention also provides an alcoholic fermentation method.

Description

technical field [0001] The invention belongs to the field of microorganisms, and in particular relates to a mutant strain of Zymomonas mobilis tolerant to high concentrations of acetic acid and its application, which can be applied to fermentation environments containing acetic acid, such as cellulose pretreatment, hydrolyzate, and kitchen waste fermentation systems. Use of bio-based products for the production of fuel ethanol. Background technique [0002] Vigorously developing renewable energy has become a global consensus on major global issues such as climate change and energy security, and cellulosic ethanol has been valued as a renewable energy. However, breaking the degradation barrier of lignocellulose is the primary obstacle for the conversion of cellulose into fuel ethanol. Currently widely used thermochemical pretreatment and other means will produce acetic acid and other by-products, which will seriously hinder the ethanol fermentation process. With the popular...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12N13/00C12N15/01C12P7/10C12P7/06C12R1/01
CPCC12N13/00C12N15/01C12P7/06C12P7/10C12N1/205C12R2001/01Y02E50/10
Inventor 何明雄吴波秦晗谭芙蓉
Owner BIOGAS SCI RES INST MIN OF AGRI
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