CN110846235A - High-temperature-resistant stress-resistant Pichia kudriavzevii high-yield ethanol and application thereof - Google Patents
High-temperature-resistant stress-resistant Pichia kudriavzevii high-yield ethanol and application thereof Download PDFInfo
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- 238000000855 fermentation Methods 0.000 claims abstract description 15
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- 239000002994 raw material Substances 0.000 claims description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 abstract description 24
- HYBBIBNJHNGZAN-UHFFFAOYSA-N furfural Chemical compound O=CC1=CC=CO1 HYBBIBNJHNGZAN-UHFFFAOYSA-N 0.000 abstract description 18
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- 235000019253 formic acid Nutrition 0.000 abstract description 7
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- 238000004321 preservation Methods 0.000 abstract description 4
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- MWOOGOJBHIARFG-UHFFFAOYSA-N vanillin Chemical compound COC1=CC(C=O)=CC=C1O MWOOGOJBHIARFG-UHFFFAOYSA-N 0.000 description 1
- FGQOOHJZONJGDT-UHFFFAOYSA-N vanillin Natural products COC1=CC(O)=CC(C=O)=C1 FGQOOHJZONJGDT-UHFFFAOYSA-N 0.000 description 1
- 235000012141 vanillin Nutrition 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
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- 239000012138 yeast extract Substances 0.000 description 1
- 239000007222 ypd medium Substances 0.000 description 1
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
- C12N1/16—Yeasts; Culture media therefor
- C12N1/165—Yeast isolates
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
- C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
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- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
- Y02E50/00—Technologies for the production of fuel of non-fossil origin
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Abstract
The invention belongs to the field of biochemical engineering, and particularly relates to a high-temperature-resistant stress-resistant high-yield pichia kudriavzevii yeast and application thereof in bioethanol production. The Pichia kudriavzevii (Pichia kudriavzevii) has the preservation number of CGMCC NO. 18379. The strain of the invention not only has high temperature tolerance, but also can tolerate various fermentation inhibitors and stress agents, such as 30mM furfural, 70mM acetic acid, 30mM formic acid and 30mM H2O2And 15% ethanol, therefore, the strain is an advantageous starting strain in the industrial production of bioethanol.
Description
Technical Field
The invention belongs to the field of biochemical engineering, and particularly relates to high-temperature-resistant stress-resistant pichia kudriavzevii for high-yield ethanol and application thereof.
Background
Bioethanol is pushed as a replacement for fossil fuels due to its advantages of sustainability, renewability, and environmental friendliness. The corn, the sugarcane, the cassava and the like used as raw materials of the bioethanol have high cost and directly compete with food supply, and the lignocellulose material is more suitable for being used as a raw material for bioethanol production.
The conversion of lignocellulosic biomass to ethanol mainly comprises three steps of pretreatment, saccharification and fermentation, wherein the pretreatment is to remove lignin components serving as a physical barrier or change the structure of the lignin components, and is a necessary condition for later-stage microbial saccharification and fermentation, byproducts such as furfural, 5-Hydroxymethylfurfural (HMF), acetate, formate, vanillin, phenolic compounds and the like are easily formed in the pretreatment process, and the process for producing ethanol through later-stage fermentation is seriously influenced.
The most commonly used ethanol production industrial strain in the prior art is Saccharomyces cerevisiae (Saccharomyces cerevisiae), but the strain can not grow and produce ethanol at the temperature of more than 30 ℃, so that ethanol can not be effectively produced by a synchronous saccharification and fermentation method.
Disclosure of Invention
The invention aims to provide a Pichia kudriavzevii (Pichia kudriavzevii) GXAS strain.
It is a further object of the present invention to provide the use of the above mentioned strains.
It is a further object of the present invention to provide a process for the industrial production of ethanol.
The strain according to the embodiment of the invention is separated from rotten fruits, is identified as the Pichia kudriavzevii (Pichia kudriavzevii) GXAS by morphological observation, genome extraction, ITS amplification and sequencing analysis, and has the preservation number of CGMCC NO. 18379.
The Pichia kudriavzevii (Pichia kudriavzevii) is deposited in the common microorganism center of China Committee for culture Collection of microorganisms (No. 3 of West Lu No.1 of the Beijing republic of the rising district, West Chen, China academy of sciences, Microbiol research institute, 100101) in 2019, 8.12.8.s.and the preservation number is CGMCC No. 18379.
The invention tests the tolerance of the Kude strain to high temperature and various stress conditions, and finds that the strain grows well at 30 ℃, 37 ℃ and 42 ℃; the ethanol yield can reach 4.56%, 4.46% and 2.96% respectively after being cultured in 10% glucose at the temperature of 30 ℃, 37 ℃ and 42 ℃; under the condition of fermentation at 42 ℃ with 20% glucose, the ethanol yield can reach 4.7% and 4.14%; in the presence of 30mM furfural, 70mM acetic acid, 50mM, 30mM H2O2Formic acid or 15% ethanol grew well in the liquid.
Based on the characteristics, the invention also provides the application of the strain, in particular the application in bioethanol production.
According to the method for industrially producing ethanol, which is disclosed by the embodiment of the invention, the Pichia kudriavzevii can be used for fermentation, lignocellulose is used as a fermentation raw material, and the fermentation is carried out at the temperature of 30-42 ℃, so that higher ethanol yield can be obtained.
The invention has the beneficial effects that:
the present invention isolates Pichia kudriavzevii (Pichia pastoris) from rotten fruits, which grows well and produces ethanol in high yield at both 37 ℃ and 42 ℃. The strain of the invention can not only have high temperature tolerance, but also can tolerate various fermentation inhibitors and stress agents, such as 30mM furfural, 70mM acetic acid, 30mM formic acid and 30mM H2O2And 15% ethanol, the strain of the invention is therefore biologicalAn advantageous starting strain in the industrial production of ethanol.
Drawings
FIG. 1 shows the results of amplification from the genome of this Kudbed strain using ITS1-ITS4 as primers;
FIG. 2 shows the growth curve of Kurthia sp.under liquid culture conditions at 30 ℃, 37 ℃ and 42 ℃;
FIG. 3 shows the results of comparison of ethanol yields of Kurd strain fermented with 10% glucose at 30 ℃, 37 ℃ and 42 ℃ for 16 hours;
FIG. 4 shows the results of comparison of ethanol yields of Kuder strains fermented at a temperature of 42 ℃ with 20% glucose for 16 hours, 40 hours and 72 hours;
FIG. 5 shows the tolerance of the curdlan strains of the invention to furfural;
FIG. 6 shows the resistance of the curdlan strains of the invention to acetic acid;
FIG. 7 shows the resistance of the curdlan strains of the invention to formic acid;
FIG. 8 shows the curdlan strain of the invention vs. H2O2(ii) a tolerance of (d);
FIG. 9 shows the tolerance of the curds strains of the invention to ethanol.
The Pichia kudriavzevii (Pichia kudriavzevii) GXAS of the present invention is deposited in the common microorganism center of the china microorganism culture preservation management committee (No. 3 of west way 1 of north chen of the yang ward area, china academy of sciences, 100101) in 2019 at 8.12.8.s.: CGMCC No. 18379.
Detailed Description
Example 1 isolation and characterization of the strains
1.1 sample Collection
Samples of yeast strains were collected from subtropical rotten fruits.
1.2 isolation of the Strain
The collected sample was inoculated into an enrichment medium YPD (containing 10% glucose, 2% peptone and 1% yeast extract), and incubated at 42 ℃ for 24 hours. mu.L of the culture was spread on a plate containing 2% glucose, 2% peptone and 2% agar and incubated at 42 ℃ for 24 hours. The single colony on the plate was inoculated into a test tube containing 3mL of YPD and incubated at 42 ℃ for 24 hours, and then the ethanol yield was measured. And (4) identifying the strain with the highest ethanol yield.
1.3 extraction of DNA from the Strain
A single colony of the strain to be identified was inoculated into 15mL of YPD and cultured for 24 hours, 3000g was centrifuged for 10 minutes, the supernatant was discarded, the pellet was resuspended and washed with sterile water, 3000g was centrifuged for 10 minutes, and the supernatant was discarded. The pellet was resuspended in 2mL buffer (20mM Tris-HCl, pH 7.5, 1.2M sorbitol, 10mM EDTA) and 100. mu.g yeast lyase was added and incubated for 30 minutes at 30 ℃. Then 200. mu.L of 10% SDS, 2mL of phenol-chloroform isoamyl alcohol were added in this order, and after thoroughly mixing, 6000g of the mixture was centrifuged for 15 minutes to separate layers. The clear aqueous phase in the supernatant was transferred to a new tube, 10. mu.L of ribonuclease was added thereto, and the temperature was maintained at 37 ℃ for 2 hours. 1mL of phenol-chloroform was added, mixed well and centrifuged at 6000g for 15 minutes. The clear aqueous phase of the supernatant was transferred to a clean tube and one-tenth volume of 3M sodium acetate and 2 volumes of absolute ethanol were added. After precipitating at-20 ℃ for 2 hours, the precipitate was centrifuged at 6000g for 15 minutes, and the supernatant was discarded. After washing the precipitate with 70% ethanol 2 times, the DNA was air-dried at room temperature.
1.4 molecular characterization of the Strain
The concentration of the extracted DNA was determined and diluted to an appropriate concentration for Polymerase Chain Reaction (PCR). The extension was performed using the universal primers ITS1/ITS4 for the fungal ribosomal rDNA region.
ITS1：TCCGTAGGTGAACCTGCGG，
ITS4：TCCTCCGCTTATTGATATGC。
The amplified product was separated by 1% agarose gel electrophoresis, and the results are shown in FIG. 1, the target fragment was recovered and purified by a DNA gel recovery kit, and then sequenced, and the strain was analyzed to be Pichia kudriavzevii (Pichia kudriavzevii) GXAS.
Example 2 examination of the characteristics of the isolated strains
The isolated strains were streaked out into single colonies on plates, and one single colony was inoculated into 3mL of YPD medium and cultured overnight. The overnight culture was taken to determine OD600 and diluted to 0.2, 200. mu.L of the culture with OD600 of 0.2 was inoculated into a 10mL fermenter, micro-oxygen supply was applied, the culture was incubated at 200rpm for various times, 1mL was taken out and the percentage concentration of ethanol was determined and calculated by HPLC.
And (3) measuring the growth curves of the strains under the stress of ethanol, formic acid, furfural, hydrogen peroxide and acetic acid at different temperatures by adopting a turbidimetric method. The method comprises the following specific steps: inoculating a single colony into 3mL YPD culture medium, culturing overnight, taking overnight culture to determine OD600, diluting with YPD culture medium without addition and with addition stress to OD600 of 0.1, placing in 96-well plate with 200 μ L per well, continuously determining for 30 hours with INFINITE 200PRO microplate reader, reading once every half hour, deducting OD value without yeast, and drawing growth curve.
The measured temperature was the growth curve of the strain at 30 ℃, 37 ℃ and 42 ℃. As shown in FIG. 2, the strain of the present invention grew well at 30 ℃, 37 ℃ and 42 ℃ and had excellent high temperature resistance.
The ethanol production of the curdlan strain of the present invention was measured under fermentation conditions of 30 ℃, 37 ℃ and 42 ℃ in a medium containing 10% glucose. As shown in FIG. 3, the ethanol yields of the strains of the present invention were 4.56%, 4.46% and 2.96% respectively when cultured at 30 deg.C, 37 deg.C and 42 deg.C for 16 hours.
The ethanol yield of the curdlan strain of the invention was determined under fermentation conditions at a temperature of 42 ℃ with 20% glucose. As shown in FIG. 4, the ethanol yields of the strains of the present invention were 4.7% and 4.14% at 40 hours and 72 hours of culture.
The growth curves of the curdlan strains of the invention were determined under liquid culture conditions containing 0, 10mM, 20mM, 30mM furfural. As shown in FIG. 5, the strain of the present invention grew well in a liquid containing 30mM furfural, and was resistant to furfural.
The growth curves of the curdlan strains of the invention were determined under liquid culture conditions containing 0, 50mM, 70mM, 100mM acetic acid. As shown in FIG. 6, the strain of the present invention grew well in a liquid containing 70mM acetic acid and was well resistant to acetic acid.
The growth curves of the curdlan strains of the invention were determined under liquid culture conditions containing 0, 10mM, 30mM, 50mM formic acid. As shown in FIG. 7, the strain of the present invention grew well in a liquid containing 50mM formic acid and was resistant to formic acid.
Determination of the Curde Strain of the invention in the presence of 0, 10mM, 20mM, 30mM H2O2The growth curve under liquid culture conditions of (1). As shown in FIG. 8, the strain of the present invention contained 30mM H2O2Is also good in the liquid of (2), for H2O2Has tolerance.
The growth curves of the curdlan strains of the invention were determined under liquid culture conditions containing 0, 5%, 10%, 15% ethanol. As shown in FIG. 9, the strain of the present invention grew well under the culture conditions containing 15% ethanol and was tolerant to ethanol.
Claims (6)
1. Pichia kudriavzevii (Pichia kudriavzevii) GXAS, wherein the accession number is CGMCC NO. 18379.
2. The use of Pichia kudriavzevii (Pichia kudriavzevii) GXAS as claimed in claim 1.
3. The use of Pichia kudriavzevii (Pichia kudriavzevii) GXAS according to claim 1 in the production of bioethanol.
4. A method for industrially producing ethanol, comprising a step of fermentation using Pichia kudriavzevii (Pichia kudriavzevii) GXAS described in claim 1.
5. The method for industrial production of ethanol according to claim 4, wherein the raw material for fermentation is lignocellulose.
6. The method for industrial production of ethanol according to claim 4, wherein the fermentation temperature is 30-42 ℃.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111635866A (en) * | 2020-06-10 | 2020-09-08 | 温州大学 | Method for inhibiting cell aggregation of pichia kudriavzevii strain, construction and application |
| CN112877375A (en) * | 2021-03-26 | 2021-06-01 | 温州大学 | Method for continuously producing xylonic acid and ethanol by using recombinant pichia pastoris fermentation |
| CN114836332A (en) * | 2022-05-27 | 2022-08-02 | 湖北稻花香酒业股份有限公司 | Pichia kudriavzevii yeast with high tolerance and low isoamylol yield and application thereof |
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| CN111635866A (en) * | 2020-06-10 | 2020-09-08 | 温州大学 | Method for inhibiting cell aggregation of pichia kudriavzevii strain, construction and application |
| CN111635866B (en) * | 2020-06-10 | 2021-11-23 | 温州大学 | Method for inhibiting cell aggregation of pichia kudriavzevii strain, construction and application |
| CN112877375A (en) * | 2021-03-26 | 2021-06-01 | 温州大学 | Method for continuously producing xylonic acid and ethanol by using recombinant pichia pastoris fermentation |
| CN114836332A (en) * | 2022-05-27 | 2022-08-02 | 湖北稻花香酒业股份有限公司 | Pichia kudriavzevii yeast with high tolerance and low isoamylol yield and application thereof |
| CN114836332B (en) * | 2022-05-27 | 2023-10-20 | 湖北稻花香酒业股份有限公司 | Pichia kudriavzevii with high tolerance and low isoamyl alcohol yield and application thereof |
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