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A kind of acetic acid and xylose co-utilization Kluyveromyces marx strain and screening method

A technology of xylose and yeast, which is applied in the field of Kluyveromyces marx strains and screening, can solve the problems of shortage and limitation of the application of cellulosic ethanol, and achieve the effects of good growth, lower production cost and higher yield

Active Publication Date: 2021-12-03
DALIAN UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the research on its inhibitor resistance is still relatively lacking, which limits its application in cellulosic ethanol production.

Method used

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  • A kind of acetic acid and xylose co-utilization Kluyveromyces marx strain and screening method
  • A kind of acetic acid and xylose co-utilization Kluyveromyces marx strain and screening method
  • A kind of acetic acid and xylose co-utilization Kluyveromyces marx strain and screening method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1: Screening of hydrolyzate-resistant yeast Kluyveromycesmarxianus DX-5

[0038] Straw steam explosion pretreatment liquid is obtained by using pulverized straw, material-water ratio 1:10, 1% sulfuric acid, 130°C, 2h. The hydrolyzate contains 30g / L xylose, 3g / L acetic acid and 0.9g / L formic acid. Add 2% glucose, 0.2% yeast extract, and 1% peptone to 10% extract hydrolyzate to make a medium for initial directed evolution. Inoculate the starting strain of Kluyveromycesmarxianus, the optical density value at 620nm (OD620) ≈ 0.3; culture at 40°C, 150rmp / min for 48 hours, when the OD620≥2.0, collect the cells; add 2% glucose to the 15% extraction hydrolyzate according to OD620 ≈ 0.3 , yeast extract 0.2%, and peptone 1% to continue directional domestication. Repeat this step to increase the hydrolyzate content in the acclimatization medium to 100%. But when OD620≥2.0, collect the cells, smear on the 100% hydrolyzate medium plate containing 1.5% agar, pick a single ...

Embodiment 2

[0039] Embodiment 2: The growth status of Kluyveromycesmarxianus DX-5 in different concentrations of acetic acid

[0040] Kluyveromycesmarxianus DX-5 was cultured in YPD medium at 40°C for 24 hours, and then inoculated into different concentrations and different types of medium according to OD620≈0.3. Kluyveromyces marxianus DX-5 can grow with different concentrations of acetic acid as the sole carbon source, and the addition of organic nitrogen sources will promote the utilization of acetic acid compared with inorganic nitrogen sources. With the increase of acetic acid concentration, the growth rate of yeast cells decreased and the depletion time of acetic acid prolonged. In the organic nitrogen source, 13g / L acetic acid can be completely consumed in 130h, and the OD620 reaches about 2. See attached for specific results figure 2 , image 3 .

Embodiment 3

[0041] Example 3: KluyveromycesmarxianusDX-5 co-utilization of xylose and acetic acid status

[0042] Kluyveromycesmarxianus DX-5 was cultured in YPD medium at 40°C for 24 hours, and then inoculated into medium containing 10g / L acetic acid and 20g / L xylose according to OD620≈0.3.

[0043] Kluyveromycesmarxianus DX-5 can utilize both acetic acid and xylose. Compared with xylose growth without acetic acid, addition of high concentration of acetic acid decreased the rate of xylose utilization but increased xylitol production. See attached for specific results Figure 4 .

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Abstract

A kind of acetic acid and xylose co-utilization Kluyveromyces marx strain strain, biological deposit number is: CGMCC No.16757. The strain is obtained from Kluyveromyces marxe after long-term directional domestication with cellulose hydrolyzate, and the steps are as follows: Pick a single colony of yeast from a YPD plate and culture overnight at 40°C; re-inoculate the collected cells to a concentration of cellulose hydrolyzate In the fresh medium of liquid, make it have an optical density value at 620nm ≈ 0.3; culture at 40°C, 150rmp / min for 24-48h, when the OD620≥2.0 (5-7 passages), collect the cells; repeat the above steps until Under the stress condition of inhibitors at this concentration, the cell growth rate was significantly improved; the concentration of the cellulose hydrolyzate was increased, and a new repeated culture was started; after continuous acclimatization, the collected cells were spread on the YPD plate containing the cellulose hydrolyzate, Cultivate at 40°C for 24-48h; pick a single colony with good growth status and culture it in YPD liquid medium at 40°C and 150rmp / min for 24-48h; store the dominant resistant clone in 30% glycerol.

Description

technical field [0001] The invention belongs to the field of genetic breeding of industrial microorganisms, and relates to a Kluyveromyces marx strain and a screening method for co-utilization of acetic acid and xylose. Background technique [0002] Lignocellulose is the most abundant renewable biomass resource, and using it as a substrate for fuel ethanol production can meet my country's demand for energy and is conducive to environmental protection. The production of fuel ethanol from lignocellulose is of great economic and social significance. Inhibitors in cellulose hydrolyzate, especially the toxic effect of acetic acid on microorganisms, is one of the main bottlenecks facing the development of cellulosic ethanol (Palmqvist, E. & Hahn-Hagerdal, B. Fermentation of lignocellulosic hydrolysates. I: inhibition and detoxification. Bioresour. Technol. 2000, 74, 17–24). Judging from the current literature reports, the acetic acid resistance of the cellulose hydrolyzate ferme...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/16C12N1/02C12R1/645
CPCC12N1/02C12N1/16C12N1/145C12R2001/645
Inventor 袁文杰杜聪相瑞娟李益民
Owner DALIAN UNIV OF TECH
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