CN111690587A - Method for centrifugally screening grease yeast strains with high oil content and application thereof - Google Patents

Method for centrifugally screening grease yeast strains with high oil content and application thereof Download PDF

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CN111690587A
CN111690587A CN201910186710.0A CN201910186710A CN111690587A CN 111690587 A CN111690587 A CN 111690587A CN 201910186710 A CN201910186710 A CN 201910186710A CN 111690587 A CN111690587 A CN 111690587A
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screening
oil content
application
high oil
strains
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CN111690587B (en
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鲍杰
赵瞭
侯伟亮
胡明珊
张建
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Shanxi Institute Of Synthetic Biology Co ltd
Cathay R&D Center Co Ltd
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East China University of Science and Technology
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N1/22—Processes using, or culture media containing, cellulose or hydrolysates thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
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    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • 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
    • Y02E50/10—Biofuels, e.g. bio-diesel

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Abstract

The invention discloses a method for centrifugally screening a strain of a fatty yeast with high oil content and application thereof, belonging to the technical field of biotechnology and microorganism. The method comprises the specific steps of culturing the grease yeast strain in lignocellulose hydrolysate, obtaining light oil lipid cells with high grease content on the upper layer through centrifugal fermentation liquor, transferring, and continuing culturing, and gradually improving the centrifugal force until the fermentation performance of the strain is stable. The new strain obtained by screening is named as filamentous spore yeast MS28, and the preservation number is CGMCC No. 14780. The method is rapid and efficient, and has important reference significance for screening all grease yeast strains.

Description

Method for centrifugally screening grease yeast strains with high oil content and application thereof
[ technical field ] A method for producing a semiconductor device
The invention relates to the technical field of biotechnology and microorganism, in particular to a method for centrifugally screening a grease yeast strain with high oil content and application thereof.
[ background of the invention ]
Grease is an important prerequisite for the production of high energy density biofuels such as biodiesel or aviation fuels. Vegetable oils from soybean rapeseed, sunflower or palm are the major sources for biodiesel and aviation fuel production. However, the tremendous biodiesel market demand requires a steady and rapid supply of substrates. Renewable lignocellulose is a very suitable source of feedstock for the production of biodiesel by fermentation.
The fermentation strain is a key factor influencing oil production, and the fermentation strain should have high tolerance of lignocellulose source inhibitor, various carbon source utilization behaviors and high oil enrichment capacity. Therefore, strain breeding is particularly important. The breeding method of the grease yeast strain mainly comprises bluish green rhzomorph staining screening, direct grease weight measurement screening, screening of a miniature bioreactor and screening of floating cells. While the artificial intelligence assisted high throughput screening is much lower than the cell number despite the increased screening efficiency, only 1% of the cells are usually screened, and the subsequent weighed oil assay greatly delays the overall screening rate.
The invention provides a method for simply and rapidly screening light oil cell strains with high oil content based on the principle of differential distribution of cell density in the centrifugal process. The method can simplify the complicated process of conventional screening and greatly improve the screening efficiency of high-yield grease cells.
[ summary of the invention ]
The invention aims to provide a method for centrifugally screening a strain of a lipidic yeast with high oil content.
The purpose of the invention is realized by the following technical scheme:
a method for centrifugally screening a fatty yeast strain with high oil content and application thereof comprise the following specific steps:
(1) inoculating parent strain at an inoculum size of 10% into 50ml 15% corn stalk hydrolysate, culturing at 30 deg.C and 180rpm for 72 hr, and adding nutrient salt component including 1.0g/L KH2PO40.5g/L yeast extract, 0.5g/LMgSO47H2O,0.22g/L (NH4)2SO4；
(2) Centrifuging 25ml of fermentation liquor for 2min under a certain centrifugal force, and sucking 5ml of supernatant of the fermentation liquor and transferring the supernatant into fresh hydrolysate for culture under the same condition;
(3) the centrifugal force is gradually increased along with the transfer until the fermentation performance of the strain is stabilized.
The inventor utilizes the principle of cell density distribution difference under the centrifugal condition to realize the screening of light cells with high oil content. The application of the simple and efficient method not only greatly improves the screening efficiency of the lipidic yeast strains, but also reduces the difficulty of screening operation.
[ description of the drawings ]
FIG. 1 is a graph of a centrifugal screening process;
FIG. 2 comparison of shake flask fermentation of parent strain and selected strain in 15% hydrolysate;
FIG. 3 shows the comparison of fermentation of the parent strain and the selected strain in 20% hydrolysate in 3L tank;
FIG. 4 comparison of 5L tank fermentations of parent and selected strains in simultaneous saccharification and co-fermentation at 30% solids.
[ detailed description ] embodiments
The following provides a method for centrifugally screening the grease yeast strain with high oil content and a specific implementation mode of the application of the method.
Example 1 Strain screening
Firstly, inoculating parent strain of Trichosporon cutaneum into 15% corn stalk hydrolysate, culturing at 30 deg.C and 180rpm for 72 hr, wherein the added nutrient salt component comprises 1.0g/L KH2PO40.5g/L yeast extract, 0.5g/L MgSO47H2O,0.22g/L(NH4)2SO4Then, the fermentation liquor is centrifuged, the centrifuged supernatant is inoculated in new hydrolysate with 10 percent of inoculum concentration and cultured under the same condition, the centrifugal force is gradually improved from 1000 × g to 12000 × g, and the light oil lipid cells are screened, finally, the obtained strain is named as filamentous spore saccharomycete (Trichosporon cutaneum) MS28 and is preserved in China general microbiological culture Collection center (CGMCC) in 2017, 10 months and 11 days, and the preservation number is CGMCC No. 14780.
Example 215% hydrolysate Shake flask fermentation
Screening strains fermentation performance was first tested on a shake flask scale. The fermentation strain is inoculated with 10% inoculum size in shake flask containing 50ml 15% hydrolysate, cultured at 30 deg.C and 180rpm for 72h, and added with nutrient salt component including 1.0g/LKH2PO40.5g/L yeast extract, 0.5g/L MgSO47H2O,0.22g/L(NH4)2SO4. The fermentation result shows that the fermentation performance of the screened strain is obviously improved, and the finally obtained oil content and the oil concentration are respectively improved by 3.2 times and 2.5 times compared with the parent strain.
Example 3L fermentation of 320% hydrolysate
Inoculating the fermentation strain with 10% inoculum size in 3L fermenter containing 1L 20% hydrolysate, and culturing at 30 deg.C under 600rpm and 1vvm aeration condition for 72 h; the pH is maintained at 5.0 during fermentation, and the nutrient salt component added during fermentation comprises 1.0g/LKH2PO40.5g/L yeast extract, 0.5g/L MgSO47H2O,0.22g/L(NH4)2SO4. The fermentation results show that compared with the parent strain, the oil content of the screened strain is increased from 17.1% to 44.5%, and the oil concentration is increased from 3.3g/L to 15.5 g/L.
Example 430% solids Simultaneous saccharification and fermentation
The pretreated and detoxified material is first pre-saccharified for 12 hr and then inoculated with 10% inoculating amount of fermenting strain. Simultaneous saccharification and fermentation was continued for 96h at 30 ℃,600rpm and 1vvm aeration; the pH is maintained at 5.0 during fermentation, and the nutrient salt component added during fermentation comprises 1.0g/L KH2PO40.5g/L yeast extract, 0.5g/L MgSO47H2O,0.22g/L(NH4)2SO4. The fermentation results show that the screened strain has stronger carbon source utilization capability compared with the parent strain, can completely utilize all sugars derived from lignocellulose, and the parent strain can only partially utilize glucose. The final screening strain obtained 34.4g/L oil accumulation while the parent strain only obtained 7.5g/L oil yield.

Claims (9)

1. A method for centrifugally screening a fatty yeast strain with high oil content and application thereof are characterized by comprising the following steps:
(1) pretreating a lignocellulose raw material, detoxifying and saccharifying to prepare a hydrolysate;
(2) inoculating grease yeast into the hydrolysate added with nutrient salt for fermentation;
(3) centrifuging the fermentation liquor, sucking supernatant containing light cells, and transferring to fresh hydrolysate for continuous culture;
(4) the centrifugal force is continuously increased along with the transfer until the fermentation performance of the strain is stable;
(5) the screened strains were evaluated for grease accumulation performance on both shake flask and fermentor scales.
2. The method for centrifugal screening of strains of oleaginous yeast with high oil content and the application thereof as claimed in claim 1, wherein in the step (1), all the lignocellulosic raw materials can be corn stalks, wheat stalks, rice straws, cotton stalks, wood chips and the like.
3. The method for centrifugally screening the strains of oleaginous yeast with high oil content and the application thereof as claimed in claim 1, wherein in the step (1), the pretreatment method is a dry dilute acid pretreatment, and the pretreatment conditions are as follows: the mass ratio of the lignocellulose solid to the dilute sulfuric acid liquid is 1:1-2:1, the dosage of the sulfuric acid is 1-10% of the dry weight of the lignocellulose raw material, the treatment temperature is 150-.
4. The method for centrifugal screening of strains of oleaginous yeast with high oil content and the application thereof as claimed in claim 1, wherein in the step (1), the detoxification method is a solid-state biological method for removing inhibitors, and the detoxification conditions are as follows: the pH value of the pretreated material is adjusted to 4.0-7.0, the water content is 50% -60%, and the resin Cladosporium Amorphotheca resinae ZN1 is inoculated in a detoxification reactor for detoxification for 12-120h under the condition of 25-35 ℃ and the ventilation capacity of 0.01-1.5 vvm.
5. The method for centrifugal screening of strains of oleaginous yeast with high oil content and the application thereof as claimed in claim 1, wherein in the step (2), the strain culture conditions are as follows: the rotation speed is 100-250rpm, the temperature is 25-40 ℃, and the culture time is 12-120 h.
6. The method for centrifugal screening of oleaginous yeast strain with high oil content and application thereof as claimed in claim 1, wherein in the step (2), the addition of nutritive salt is 0-5g/L KH2PO40-5g/L yeast extract, 0-5g/L MgSO47H2O,0-5g/L(NH4)2SO4。
7. The method for centrifugal screening of strains of oleaginous yeast with high oil content and the application thereof as claimed in claim 1, wherein in the step (3), the amount of inoculation of upper light cells into fresh hydrolysate is 1-30%.
8. The method for centrifugal screening of strains of oleaginous yeast with high oil content and the application thereof according to claim 1, wherein in the step (4), the centrifugal force is 30000 Xg.
9. The method for centrifugal screening of strains of oleaginous yeast with high oil content and the application thereof as claimed in claim 1, wherein in the step (4), the new strains obtained by screening are preserved in China general microbiological culture Collection center (CGMCC) in 2017, 10 and 11 months, and the preservation number is CGMCC No. 14780.
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