CN115948262A - Separated high-activity stress-resistant yeast and ethanol fermentation application thereof - Google Patents
Separated high-activity stress-resistant yeast and ethanol fermentation application thereof Download PDFInfo
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- 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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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses a separated high-activity stress-resistant yeast and an ethanol fermentation application thereof, belonging to the technical field of biology. The Saccharomyces cerevisiae SG provided by the invention is separated from a storage tank sediment sample of an ethanol fermentation plant under the environment of high temperature, anaerobism and multiple stress of inhibitors, and is preserved in a China center for type culture Collection (CCTCC M20221770) at 11 months and 11 days in 2022 years. The Saccharomyces cerevisiae (Saccharomyces cerevisiae) SG can effectively solve the problems of high sugar, high permeability, high temperature and multiple stress of inhibitors in the anaerobic ethanol fermentation process, has good characteristics of strain stress resistance, cell growth, substrate utilization and ethanol synthesis, and can improve the concentration of thalli, the conversion rate of sugar alcohol and the production intensity of ethanol.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a separated high-activity stress-resistant yeast and an ethanol fermentation application thereof.
Background
China is the largest crude oil import country in the world at present, the energy structure is mainly fossil energy, the crude oil import quantity and the external dependence degree are high for a long time, one-time energy consumption causes huge carbon emission pressure, and the key technology research and development and equipment manufacturing such as cellulose ethanol are required to be accelerated. The advanced biofuel is actively promoted, and the fossil energy is replaced, popularized and applied in key fields of municipal administration, traffic and the like, so that the transformation from the fossil energy to green low-carbon renewable energy is promoted. Meanwhile, according to the opinions on the mechanism and policy measures of the improved energy green low-carbon transformation system which are jointly issued recently by the State development Commission and the State energy agency, the key technologies of promoting the energy green low-carbon transformation, developing the advanced biological liquid fuels such as bioethanol and the like and accelerating the non-grain biological fuels such as cellulose and the like are required to collaboratively overcome. Therefore, the bioethanol is used as a low-pollution renewable alternative energy source, has obvious energy benefit and carbon emission reduction benefit, and has great significance for ensuring sustainable development, energy safety and environmental friendliness.
Saccharomyces cerevisiae is used as a main production strain for bioethanol fermentation, glucose can be used for anaerobic fermentation to synthesize an ethanol product, in the process, carbon source substrates such as glucose and the like form ethanol through glycolysis by the catalytic action of ethanol dehydrogenase, and the Saccharomyces cerevisiae cannot perform aerobic respiration by taking oxygen in the anaerobic fermentation process, so that in the anaerobic ethanol fermentation process, for most wild-type or naturally-existing Saccharomyces cerevisiae, the thallus growth concentration or cell density is low, and the stress resistance of thallus cells to high-temperature high-sugar and inhibitors is poor, thereby further influencing the carbon source utilization, ethanol synthesis activity and fermentation production efficiency.
At present, the saccharomyces cerevisiae is generally subjected to anaerobic ethanol fermentation at the temperature of 30-36 ℃, and is often subjected to stronger metabolic activity inhibition in hydrolysate containing toxic inhibitors, high-temperature environment, high-sugar environment or high-permeability environment. Therefore, the problems of high sugar, high permeability, high temperature and multiple stress of inhibitors of the yeast strains in the anaerobic ethanol fermentation process are effectively solved, the stress resistance, cell growth, substrate utilization and ethanol synthesis activity of the strains are synergistically enhanced, the cell concentration of the strains, the sugar-alcohol conversion rate and the ethanol production strength are improved, and the method is of great importance for improving the yield and income of bioethanol fermentation.
Disclosure of Invention
The invention provides a separated high-activity stress-resistant yeast and an ethanol fermentation application thereof, in order to effectively solve the problems of high sugar, high permeability, high temperature and multiple stress of an inhibitor of a yeast strain in the ethanol fermentation process, synergistically strengthen the stress resistance, cell growth, substrate utilization and ethanol synthesis characteristics of the strain and improve the thallus concentration, sugar alcohol conversion rate and ethanol production strength.
The invention provides a high-activity stress-resistant yeast, which is separated from the anaerobic environment of the bottom sludge of a storage tank of an ethanol fermentation plant under the environment of high temperature, anaerobism and multiple stress of inhibitors, and is preserved in China center for type culture collection (address: eight-way 299 of Wuchang district, wuhan city, hubei province, zip code 430072) at 11 months and 11 days in 2022, and is named as Saccharomyces cerevisiae (Saccharomyces cerevisiae) SG, wherein the preservation number is as follows: CCTCC M20221770.
The second aspect of the invention provides an application of Saccharomyces cerevisiae (Saccharomyces cerevisiae) SG in anaerobic ethanol fermentation under high-temperature environment and high-sugar environment by utilizing a synthetic medium and a toxic hydrolysate medium.
In the application, the concentration of fermentation sugar in the used synthetic culture medium and the toxic hydrolysate culture medium is 20-240g/L.
Furthermore, the used toxic hydrolysate is supernatant containing fermentation sugar and inhibitor obtained by pretreating straw raw materials with dilute acid and performing solid-liquid separation, the used high-temperature environment temperature is 36-45 ℃, and the used high-sugar environment sugar concentration is 100-240g/L.
Furthermore, the high temperature environment temperature is 36-45 ℃, and the high sugar environment sugar concentration is 100-240g/L.
The third aspect of the invention provides an application method of high-activity stress-resistant Saccharomyces cerevisiae (Saccharomyces cerevisiae) SG in ethanol fermentation, which comprises the following steps:
(1) Diluting and coating a Saccharomyces cerevisiae (Saccharomyces cerevisiae) SG preservation solution on a YPD solid culture medium under an aseptic condition, and performing static culture at a constant temperature of 30-45 ℃ for 12-48h to obtain a single colony for liquid activation culture;
(2) Under the aseptic condition, inoculating the single colony in the step (1) into a YPD liquid culture medium, carrying out shake culture at 30-45 ℃ and 150-200rpm for 24-48h, and preparing an activated bacterial liquid for anaerobic ethanol fermentation culture;
(3) Under the aseptic condition, inoculating the activated bacterium liquid obtained in the step (2) into a liquid synthetic culture medium or a toxic hydrolysate culture medium according to the inoculation amount of 5-10%, performing closed anaerobic ethanol fermentation culture at the temperature of 30-45 ℃ and the rpm of 150-200 for 24-72h.
Furthermore, the nutrient components of the synthetic culture medium or hydrolysate culture medium comprise 20-240g/L of glucose, 6-30g/L of yeast extract, 6-15g/L of peptone, 0.8-4g/L of ammonium sulfate, 0.8-4g/L of potassium dihydrogen phosphate, 0.5-2.5g/L of urea, 0.5-1.5g/L of sodium glutamate, 0.5-1.0g/L of magnesium sulfate heptahydrate, 0.001-0.01g/L of manganese sulfate monohydrate, 0.05-0.5g/L of zinc sulfate heptahydrate, 0.001-0.01g/L of copper sulfate pentahydrate, 0.01-0.1g/L of calcium chloride, 0.01-0.1g/L of ferric chloride hexahydrate, 0.001-0.01g/L of potassium iodide, 0.05-0.15g/L of inositol, 0.3236 z3236-0.1 g/L of vitamin B, 0.01-0.1g/L of sodium chloride hexahydrate, 0.01-0.1g/L of potassium iodide, 0.05-0.01-0.63 g/L of natural amino-0.01-0.5 g/L of sulfuric acid sulfate, and the balance of sodium sulfate.
The invention has the beneficial effects that: the high-activity stress-resistant yeast, namely Saccharomyces cerevisiae SG, is a storage tank sediment sample of an ethanol fermentation plant separated in a high-temperature, anaerobic and inhibitor multi-stress environment, has good strain stress resistance, cell growth, substrate utilization and ethanol synthesis characteristics by utilizing a synthetic culture medium and a toxic hydrolysis liquid culture medium in a high-temperature environment and a high-sugar environment, is improved by 8.6-100 percent compared with the strain concentration of the traditional ethanol production strains such as Saccharomyces cerevisiae EB, 4126, 6525 and the like, is stable in sugar-alcohol conversion rate of 0.41-0.45g/g, and has ethanol production strength of 1.9-4.0g/L/h. The separated high-activity stress-resistant yeast and the ethanol fermentation application thereof provided by the invention enrich the excellent strain resources and varieties for ethanol production, effectively solve the problems of high sugar, high permeability, high temperature and multiple stress of inhibitors in the ethanol fermentation process of the yeast strain, improve the indexes of anaerobic ethanol fermentation capacity, efficiency and the like, and synergistically strengthen the stress resistance, cell growth, substrate utilization and ethanol synthesis characteristics of the yeast strain, improve the strain concentration, sugar-alcohol conversion rate and ethanol production strength in the anaerobic ethanol fermentation process of the yeast strain, and provide a new idea for the development of biomass energy sources such as ethanol and the like.
Drawings
FIG. 1 is a schematic diagram showing the morphology of a Saccharomyces cerevisiae (Saccharomyces cerevisiae) SG colony under culture conditions of 40 ℃;
FIGS. 2 (a) to 2 (c) are schematic diagrams showing the comparison of anaerobic ethanol fermentation of Saccharomyces cerevisiae EB, 4126, 6525, SG at 30 ℃ using synthetic medium (containing 100g/L glucose);
FIGS. 3 (a) to 3 (c) are schematic diagrams showing the anaerobic ethanol fermentation of Saccharomyces cerevisiae EB, 4126, 6525, SG at 30 ℃ by using toxic hydrolysate (containing 100g/L glucose);
FIGS. 4 (a) to 4 (c) are schematic diagrams showing the anaerobic ethanol fermentation of Saccharomyces cerevisiae EB, 4126, 6525, SG with toxic hydrolysate (containing 100g/L glucose) at 40 ℃;
FIGS. 5 (a) to 5 (c) are schematic diagrams showing the anaerobic ethanol fermentation of Saccharomyces cerevisiae SG at 38 ℃ by using toxic hydrolysate (containing 160, 200 and 240g/L glucose).
Detailed Description
The present invention is further illustrated by the following examples.
Example 1: isolation of Saccharomyces cerevisiae SG
The high-activity anti-adversity yeast, namely Saccharomyces cerevisiae SG, is obtained by separating a bottom sediment sample of a storage tank of an ethanol fermentation plant under the environment of high temperature, anaerobism and multiple stress of inhibitors, and performing gradient dilution and coating on the collected sample and then separating the sample. The specific separation and screening operation steps are as follows:
a. collecting a bottom mud sample of a storage tank of an ethanol fermentation plant, wherein the sampling environment is anaerobic, the temperature is 36-45 ℃, the sample is rich in various inhibitors, the sampling amount is 1mL or 1g, adding the sample into a triangular shake flask containing 99mL of sterile water, and carrying out shake culture for 60-120min at the temperature of 30-45 ℃ and the speed of 150-200 rpm.
b. Gradient dilution with pipettor (10) -2 、10 -3 、10 -4 ) Coating 100 mu L of diluent on a YPD solid culture medium, coating 10 culture dishes at each dilution concentration, placing 5 culture dishes in a group in an incubator at 30-45 ℃ for anaerobic culture for 24-72h, observing the colony formation condition, selecting different single colonies, carrying out dilution and streak separation culture, and transferring to the YPD solid culture medium for streak purification and preservation.
The YPD solid medium consists of (g/L): peptone 20, yeast extract 10, glucose 20, agar 15. The balance of water is added to 1000mL, the pH is natural, and the high-pressure moist heat sterilization is carried out for 15min at 121 ℃.
Wherein, anaerobic culture is carried out for 24-72h in an incubator at 30-45 ℃ on all culture dishes coated according to different dilution concentrations, and a plurality of single colonies growing out fastest under different culture conditions are respectively purified and preserved.
Example 2: identification of Saccharomyces cerevisiae SG
The strain morphology is characterized as follows: after culturing on YPD solid medium at 30-45 deg.C for 24-72h, the formed single colony is round, milky white, smooth and glossy in surface, and regular in colony edge. According to the manual of fungus identification, a single colony strain is judged to have typical fungus morphological characteristics.
Extracting the genomic DNA of the separated and purified strain, performing 18s rDNA sequencing analysis (the sequencing result is shown as SEQ ID NO. 1) by the company Limited in the biological engineering (Shanghai) through BLAST homologous comparison in an NCBI database, selecting Saccharomyces cerevisiae and a sequence thereof with higher homology, performing multi-sequence comparison analysis by ClustalX1.81 software, and preliminarily identifying and separating the purified strain into Saccharomyces cerevisiae (Saccharomyces cerevisiae) which is preserved in a China center for type culture preservation 11/2022 and named as Saccharomyces cerevisiae (Saccharomyces cerevisiae) SG, wherein the description is referred to as Saccharomyces cerevisiae SG or Sc SG, the preservation number is CCTCC M20221770, and the preservation unit address is Wuhan Wuchang region eight-one-way 299 number in Hubei province. As shown in FIG. 1, the colony morphology of Saccharomyces cerevisiae (Saccharomyces cerevisiae) SG was observed under the culture condition of 40 ℃.
SEQ ID NO.1：
CCATACTCCCCCCAGACCCAAAGACTTTGATTTCTCGTAAGGTGCCGAGTGGGTCATTAAAAAAACACCACCCGATCCCTAGTCGGCATAGTTTATGGTTAAGACTACGACGGTATCTGATCATCTTCGATCCCCTAACTTTCGTTCTTGATTAATGAAAACGTCCTTGGCAAATGCTTTCGCAGTAGTTAGTCTTCAATAAATCCAAGAATTTCACCTCTGACAATTGAATACTGATGCCCCCGACCGTCCCTATTAATCATTACGATGGTCCTAGAAACCAACAAAATAGAACCAAACGGCCTATTCTATTATTCCATGCTAATATATTCGAGCAATACGCCTGCTTTGAACACTCTAATTTTTTCAAAGTAAAAGTCCTGGTTCGCCAAGAGCCACAAGGACTCAAGGTTAGCCAGAAGGAAAGGCCCCGTTGGAAATCCAGTACACGAAAAAATCGGACCGGCCAACAGGGCCCAAAGTTCAACTACGAGCTTTTTAACTGCAACAACTTTAATATACGCTATTGGAGCTGGAATTACCGCGGCTGCTGGCACCAGACTTGCCCTCCAATTGTTCCTCGGTAAGGTATTTACATTGTACTCATTCCAATTACAAGACCCGAATGGGCCCTGTATCGTTATTTATTGTCACTACCTCCCTGAATTAGGATTGGGTAATTTGCGCGCCTGCTGCCTTCCTTGGATGTGGTAGCCGTTTCTCAGGCTCCCTCTCCGGAATCGAACCCTTATTCCCCGTTACCCGTTTAAACCATGGTAGGCCACTATCCTACCATCGAAAGTTGATAGGGCAGAAATTTGAATGAACCATCGCCAGCACAAGGCCATGCGATTCGAAAAGTTATTATGAATCATCAAAGAGTCCGAAGACATTGATTTTTTATCTAATAAATACATCTCTTCCAAAGGGTCGAGATTTTAAGCATGTATTAGCTCTAGAATTACCACAGTTATACCATGTAGTAAAGGGAACTATCAAATAAACGATAACTGATTTAATGAGCCATTCGCAGTTTCACTGTAAAAATTGCTTATACTTAGAACATGCATGGCTTAATCTTTGTAGACA
Example 3: anaerobic ethanol fermentation of Saccharomyces cerevisiae at 30 ℃ Using synthetic Medium (containing 100g/L glucose 1. Saccharomyces cerevisiae SG ethanol fermentation
The saccharomyces cerevisiae SG obtained in the embodiment 2 is used as an experimental group strain, and is sequentially subjected to plate culture, activation culture and fermentation culture experiments at the temperature of 30 ℃, and the specific operation steps are as follows:
a. diluting and coating the saccharomyces cerevisiae SG preservation solution on a YPD solid culture medium under an aseptic condition, and performing static culture at a constant temperature of 30 ℃ for 12-48h to obtain a single colony for liquid activated culture;
b. under the aseptic condition, inoculating the saccharomyces cerevisiae SG single colony in the step a into a YPD liquid culture medium, carrying out shaking culture at 30 ℃ and 150rpm for 24h, and preparing an activated bacterial liquid for fermentation culture;
c. and c, inoculating the saccharomyces cerevisiae SG activated bacterial liquid obtained in the step b into a liquid synthetic culture medium according to the inoculation amount of 10% under the aseptic condition, performing closed anaerobic ethanol fermentation culture at the temperature of 30 ℃ and the rpm of 150 for 24 hours.
The synthetic medium components used comprise 100g/L glucose, 6g/L yeast extract, 6g/L peptone, 1.5g/L ammonium sulfate, 1.5g/L potassium dihydrogen phosphate, 1g/L urea, 1g/L sodium glutamate, 0.7g/L magnesium sulfate heptahydrate, 0.005g/L manganese sulfate monohydrate, 0.4g/L zinc sulfate heptahydrate, 0.003g/L copper sulfate pentahydrate, 0.02g/L calcium chloride, 0.02g/L ferric chloride hexahydrate, 0.001g/L potassium iodide, 0.09g/L inositol, 0.07g/L vitamin B, 0.3g/L biotin, 0.04g/L nicotinic acid, 0.01g/L p-aminobenzoic acid, 0.02g/L HCl-thiamine, 0.04g/L HCl-pyridoxal, the balance water, and natural pH.
2. Saccharomyces cerevisiae EB, 4126, 6525 ethanol fermentation (control)
Saccharomyces cerevisiae EB, 4126 and 6525 (hereinafter referred to as Saccharomyces cerevisiae EB, 4126 and 6525 or Sc EB, sc 4126 and Sc 6525) are taken as control strains, are stored in a laboratory, and are respectively subjected to plate culture, activation culture and fermentation culture experiments in sequence at the temperature of 30 ℃, and the specific operation steps are as follows:
a. under aseptic condition, respectively diluting and coating the preservation solutions of saccharomyces cerevisiae EB, 4126 and 6525 on a YPD solid culture medium, and standing and culturing at constant temperature of 30 ℃ for 12-48h to obtain single colonies for liquid activation culture;
b. respectively inoculating single colonies of the saccharomyces cerevisiae EB, 4126 and 6525 in the step a into a YPD liquid culture medium under the aseptic condition, carrying out shaking culture at 30 ℃ and 150rpm for 24 hours, and preparing activated bacterial liquid for fermentation culture;
c. under the aseptic condition, respectively inoculating the activated bacteria liquid of the saccharomyces cerevisiae EB, 4126 and 6525 in the step b into a liquid synthetic culture medium according to the inoculation amount of 10%, performing closed anaerobic ethanol fermentation culture at 30 ℃ and 150rpm for 24 hours.
The synthetic medium ingredients used comprised 100g/L glucose, 6g/L yeast extract, 6g/L peptone, 1.5g/L ammonium sulfate, 1.5g/L potassium dihydrogen phosphate, 1g/L urea, 1g/L sodium glutamate, 0.7g/L magnesium sulfate heptahydrate, 0.005g/L manganese sulfate monohydrate, 0.4g/L zinc sulfate heptahydrate, 0.003g/L copper sulfate pentahydrate, 0.02g/L calcium chloride, 0.02g/L ferric chloride hexahydrate, 0.001g/L potassium iodide, 0.09g/L inositol, 0.07g/L vitamin B5, 0.3g/L biotin, 0.04g/L nicotinic acid, 0.01g/L para-aminobenzoic acid, 0.02g/L HCl-thiamine, 0.04g/L HCl-pyridoxal, and the balance water, natural pH.
As shown in FIGS. 2 (a) to 2 (c), the anaerobic ethanol fermentations of Saccharomyces cerevisiae EB, 4126, 6525, SG at 30 ℃ in synthetic medium (containing 100g/L glucose) were compared. The result shows that the maximum value of the concentration OD600 of the saccharomyces cerevisiae SG bacteria is 7.6, which is respectively improved by 10.1%, 8.6% and 8.6% compared with saccharomyces cerevisiae EB, 4126 and 6525. The highest ethanol concentration of 43g/L, the sugar-alcohol conversion rate of 0.43g/g and the ethanol production intensity of 3.58g/L/h can be achieved after the saccharomyces cerevisiae SG is fermented for 12 h; correspondingly, after 18 hours of fermentation, the highest ethanol concentration of the saccharomyces cerevisiae EB, the saccharomyces cerevisiae 4126 and the saccharomyces cerevisiae 6525 is 41.3 to 41.5g/L, the sugar-alcohol conversion rate is 0.41 to 0.42g/g, and the ethanol production intensity is 2.29 to 2.31g/L/h. It can be seen that the saccharomyces cerevisiae SG of the present invention has higher logarithmic growth, substrate consumption and ethanol synthesis activity.
Example 4: anaerobic ethanol fermentation of Saccharomyces cerevisiae with toxic hydrolysate (containing 100g/L glucose) at 30 deg.C 1, ethanol fermentation of Saccharomyces cerevisiae SG
The saccharomyces cerevisiae SG obtained in the embodiment 2 is used as an experimental group strain, and is subjected to plate culture, activation culture and fermentation culture experiments in sequence at the temperature of 30 ℃, and the specific operation steps are as follows:
a. diluting and coating the saccharomyces cerevisiae SG preservation solution on a YPD solid culture medium under an aseptic condition, and performing static culture at a constant temperature of 30 ℃ for 12-48h to obtain a single colony for liquid activated culture;
b. under the aseptic condition, inoculating the saccharomyces cerevisiae SG single colony in the step a into a YPD liquid culture medium, carrying out shaking culture at 30 ℃ and 150rpm for 24h, and preparing an activated bacterial liquid for fermentation culture;
c. and c, inoculating the saccharomyces cerevisiae SG activated bacterial liquid obtained in the step b into a toxic hydrolysate culture medium according to the inoculation amount of 10% under the aseptic condition, performing closed anaerobic ethanol fermentation culture at the temperature of 30 ℃ and the rpm of 150 for 24 hours.
The culture medium components of the used toxic hydrolysate comprise 100g/L of glucose, 6g/L of yeast extract, 6g/L of peptone, 1.5g/L of ammonium sulfate, 1.5g/L of potassium dihydrogen phosphate, 1g/L of urea, 1g/L of sodium glutamate, 0.7g/L of magnesium sulfate heptahydrate, 0.005g/L of manganese sulfate monohydrate, 0.4g/L of zinc sulfate heptahydrate, 0.003g/L of copper sulfate pentahydrate, 0.02g/L of calcium chloride, 0.02g/L of ferric chloride hexahydrate, 0.001g/L of potassium iodide, 0.09g/L of inositol, 0.07g/L of vitamin B, 0.3g/L of biotin, 0.04g/L of nicotinic acid, 0.01g/L of p-aminobenzoic acid, 0.02g/L of HCl-thiamine, 0.04g/L of HCl-pyridoxal, and the balance of acidolysis solution and natural pH.
2. Saccharomyces cerevisiae EB, 4126, 6525 ethanol fermentation (control)
Saccharomyces cerevisiae EB, 4126 and 6525 (hereinafter referred to as Saccharomyces cerevisiae EB, 4126 and 6525 or Sc EB, sc 4126 and Sc 6525) are taken as control strains, are stored in a laboratory, and are respectively subjected to plate culture, activation culture and fermentation culture experiments in sequence at the temperature of 30 ℃, and the specific operation steps are as follows:
a. respectively diluting and coating the preservation solutions of saccharomyces cerevisiae EB, 4126 and 6525 on a YPD solid culture medium under the aseptic condition, and standing and culturing at the constant temperature of 30 ℃ for 12-48h to obtain a single colony for liquid activation culture;
b. respectively inoculating single colonies of the saccharomyces cerevisiae EB, 4126 and 6525 in the step a into a YPD liquid culture medium under the aseptic condition, carrying out shaking culture at 30 ℃ and 150rpm for 24 hours, and preparing activated bacterial liquid for fermentation culture;
c. under the aseptic condition, respectively inoculating the activated bacteria liquid of the saccharomyces cerevisiae EB, 4126 and 6525 in the step b into a toxic hydrolysate culture medium according to the inoculation amount of 10%, performing closed anaerobic ethanol fermentation culture at 30 ℃ and 150rpm for 24 hours.
The culture medium components of the used toxic hydrolysate comprise 100g/L of glucose, 6g/L of yeast extract, 6g/L of peptone, 1.5g/L of ammonium sulfate, 1.5g/L of potassium dihydrogen phosphate, 1g/L of urea, 1g/L of sodium glutamate, 0.7g/L of magnesium sulfate heptahydrate, 0.005g/L of manganese sulfate monohydrate, 0.4g/L of zinc sulfate heptahydrate, 0.003g/L of copper sulfate pentahydrate, 0.02g/L of calcium chloride, 0.02g/L of ferric chloride hexahydrate, 0.001g/L of potassium iodide, 0.09g/L of inositol, 0.07g/L of vitamin B, 0.3g/L of biotin, 0.04g/L of nicotinic acid, 0.01g/L of p-aminobenzoic acid, 0.02g/L of HCl-thiamine, 0.04g/L of HCl-pyridoxal, and the balance of acidolysis solution and natural pH.
As shown in FIGS. 3 (a) to 3 (c), saccharomyces cerevisiae EB, 4126, 6525, SG were fermented at 30 ℃ in anaerobic ethanol using a toxic hydrolysate (containing 100g/L glucose). The result shows that the maximum value of the concentration OD600 of the saccharomyces cerevisiae SG thalli is 6.3, which is respectively improved by 70.3%, 65.8% and 75.0% compared with saccharomyces cerevisiae EB, 4126 and 6525. The fermentation time of saccharomyces cerevisiae SG reaches higher ethanol concentration of 42.8g/L and ethanol production intensity of 3.57g/L/h after 12h, while the ethanol concentrations of saccharomyces cerevisiae EB, saccharomyces cerevisiae 4126 and saccharomyces cerevisiae 6525 are only 24, 26.5 and 21.5g/L at the same time, and the ethanol production intensity is 1.79 to 2.21g/L/h. The saccharomyces cerevisiae SG is rapidly fermented for 12 hours to exhaust all glucose, and the conversion rate of sugar alcohol is 0.45g/g; saccharomyces cerevisiae EB, 4126, 6525 was slowly fermented for 24h to deplete all glucose and to achieve a sugar alcohol conversion of 0.44g/g. It can be seen that the saccharomyces cerevisiae SG of the present invention has higher logarithmic growth, substrate consumption and ethanol synthesis activity.
Example 5: anaerobic ethanol fermentation of Saccharomyces cerevisiae at 40 ℃ Using synthetic Medium (containing 100g/L glucose 1. Saccharomyces cerevisiae SG ethanol fermentation
The saccharomyces cerevisiae SG obtained in the embodiment 2 is used as an experimental group strain, and is sequentially subjected to plate culture, activation culture and fermentation culture experiments at the temperature of 30-40 ℃, and the specific operation steps are as follows:
a. diluting and coating the saccharomyces cerevisiae SG preservation solution on a YPD solid culture medium under an aseptic condition, and performing static culture at a constant temperature of 30 ℃ for 12-48h to obtain a single colony for liquid activated culture;
b. under the aseptic condition, inoculating the saccharomyces cerevisiae SG single colony in the step a into a YPD liquid culture medium, carrying out shaking culture at 30 ℃ and 150rpm for 24h, and preparing an activated bacterial liquid for fermentation culture;
c. under the aseptic condition, inoculating the saccharomyces cerevisiae SG activated bacterial liquid in the step b into a toxic hydrolysate culture medium according to the inoculation amount of 10%, and carrying out closed anaerobic ethanol fermentation culture for 24 hours at 40 ℃ and 150 rpm.
2. The medium components used for the toxic hydrolysate comprise 100g/L glucose, 6g/L yeast extract, 6g/L peptone, 1.5g/L ammonium sulfate, 1.5g/L potassium dihydrogen phosphate, 1g/L urea, 1g/L sodium glutamate, 0.7g/L magnesium sulfate heptahydrate, 0.005g/L manganese sulfate monohydrate, 0.4g/L zinc sulfate heptahydrate, 0.003g/L copper sulfate pentahydrate, 0.02g/L calcium chloride, 0.02g/L ferric chloride hexahydrate, 0.001g/L potassium iodide, 0.09g/L inositol, 0.07g/L vitamin B, 0.3g/L biotin, 0.04g/L nicotinic acid, 0.01g/L p-aminobenzoic acid, 0.02g/L HCl-thiamine, 0.04g/L HCl-pyridoxal, and the balance acidolysis solution and natural pH. Saccharomyces cerevisiae EB, 4126, 6525 ethanol fermentation (control)
Saccharomyces cerevisiae EB, 4126 and 6525 (hereinafter referred to as Saccharomyces cerevisiae EB, 4126 and 6525 or Sc EB, sc 4126 and Sc 6525) are taken as control strains, are stored in a laboratory, and are respectively subjected to plate culture, activation culture and fermentation culture experiments in sequence at the temperature of 30-40 ℃, and the specific operation steps are as follows:
a. under aseptic condition, respectively diluting and coating the preservation solutions of saccharomyces cerevisiae EB, 4126 and 6525 on a YPD solid culture medium, and standing and culturing at constant temperature of 30 ℃ for 12-48h to obtain single colonies for liquid activation culture;
b. respectively inoculating single colonies of the saccharomyces cerevisiae EB, 4126 and 6525 in the step a into a YPD liquid culture medium under the aseptic condition, carrying out shaking culture at 30 ℃ and 150rpm for 24 hours, and preparing activated bacterial liquid for fermentation culture;
c. under the aseptic condition, respectively inoculating the activated bacterium liquid of the saccharomyces cerevisiae EB, 4126 and 6525 in the step b into a toxic hydrolysate culture medium according to the inoculation amount of 10 percent, fermenting and culturing for 24 hours at 40 ℃ and 150rpm in a closed anaerobic ethanol manner.
The culture medium components of the used toxic hydrolysate comprise 100g/L of glucose, 6g/L of yeast extract, 6g/L of peptone, 1.5g/L of ammonium sulfate, 1.5g/L of potassium dihydrogen phosphate, 1g/L of urea, 1g/L of sodium glutamate, 0.7g/L of magnesium sulfate heptahydrate, 0.005g/L of manganese sulfate monohydrate, 0.4g/L of zinc sulfate heptahydrate, 0.003g/L of copper sulfate pentahydrate, 0.02g/L of calcium chloride, 0.02g/L of ferric chloride hexahydrate, 0.001g/L of potassium iodide, 0.09g/L of inositol, 0.07g/L of vitamin B, 0.3g/L of biotin, 0.04g/L of nicotinic acid, 0.01g/L of p-aminobenzoic acid, 0.02g/L of HCl-thiamine, 0.04g/L of HCl-pyridoxal, and the balance of acidolysis solution and natural pH. As shown in FIGS. 4 (a) to 4 (c), saccharomyces cerevisiae EB, 4126, 6525, SG were fermented at 40 ℃ in anaerobic ethanol using a toxic hydrolysate (containing 100g/L glucose). The result shows that the maximum value of the concentration OD600 of the saccharomyces cerevisiae SG thalli is 4.0, which is respectively improved by 100%, 100% and 60% compared with saccharomyces cerevisiae EB, 4126 and 6525. The saccharomyces cerevisiae SG is fermented for 12 hours to quickly consume nearly 90g/L of glucose, and the saccharomyces cerevisiae EB, 4126 and 6525 only slowly consume 62, 67.5 and 55.5g/L of glucose at the same time; at the end of fermentation, the ethanol concentration of saccharomyces cerevisiae SG is 45.4g/L, the sugar alcohol conversion rate is 0.45g/g, and the ethanol concentrations of saccharomyces cerevisiae EB, 4126 and 6525 are 41.8-42.4g/L respectively, and the sugar alcohol conversion rate is 0.42g/g. It can be seen that the saccharomyces cerevisiae SG of the present invention has higher logarithmic growth, substrate consumption and ethanol synthesis activity.
Example 6: anaerobic ethanol fermentation of Saccharomyces cerevisiae SG with toxic hydrolysate (containing 160, 200 and 240g/L glucose) at 40 deg.C
The saccharomyces cerevisiae SG obtained in the embodiment 2 is used as an experimental group strain, and is sequentially subjected to plate culture, activation culture and fermentation culture experiments at the temperature of 30-40 ℃, and the specific operation steps are as follows:
a. under the aseptic condition, diluting and coating the saccharomyces cerevisiae SG preservation solution on a YPD solid culture medium, and standing and culturing for 12-48h at the constant temperature of 30 ℃ to obtain a single colony for liquid activated culture;
b. under the aseptic condition, inoculating the single saccharomyces cerevisiae SG colony obtained in the step a into a YPD liquid culture medium, performing shaking culture at 30 ℃ and 150rpm for 24 hours, and preparing activated bacterial liquid for fermentation culture;
c. and c, inoculating the saccharomyces cerevisiae SG activated bacteria liquid obtained in the step b into a toxic hydrolysate culture medium according to the inoculation amount of 10% under an aseptic condition, and performing closed anaerobic ethanol fermentation culture for 48 hours at 38 ℃ at 150 rpm.
The medium components of the used toxic hydrolysate comprise 160g/L or 200g/L or 240g/L glucose, 10g/L or 20g/L or 30g/L yeast extract, 9g/L or 12g/L or 15g/L peptone, 2.25g/L or 3g/L ammonium sulfate, 2.25g/L or 3g/L potassium dihydrogen phosphate, 3g/L or 3.75g/L urea, 1g/L sodium glutamate, 0.7g/L magnesium sulfate heptahydrate, 0.005g/L manganese sulfate monohydrate, 0.4g/L zinc sulfate heptahydrate, 0.003g/L copper sulfate pentahydrate, 0.02g/L calcium chloride, 0.02g/L ferric chloride hexahydrate, 0.001g/L potassium iodide, 0.09g/L inositol, 0.07g/L vitamin B5, 0.3g/L biotin, 0.04g/L niacin, 0.01g/L para aminobenzoic acid, 0.04g/L picolinic acid, 0.04g/L HCl and the balance of natural amino-benzene amino-acetic acid. As shown in FIGS. 5 (a) to 5 (c), the anaerobic ethanol fermentation of Saccharomyces cerevisiae SG at 40 ℃ using toxic hydrolysate (containing 160, 200, 240g/L glucose) was compared. The result shows that the maximum value of the concentration OD600 of the saccharomyces cerevisiae SG bacterial body is still 3.3-4.0, nearly 220g/L glucose can be consumed at the highest time after 48 hours of fermentation, the highest concentration of fermentation ethanol is 90g/L, and the conversion rate of the sugar alcohol is 0.45, 0.43 and 0.41g/g in sequence. Therefore, the saccharomyces cerevisiae SG has higher logarithmic growth, substrate consumption and ethanol synthesis activity under the conditions of inhibitor stress and high temperature and sugar, and keeps good ethanol fermentation performance.
Claims (10)
1. A high-activity stress-resistant yeast is deposited in China center for type culture Collection (CGMCC) at 11 months and 11 days 2022, is named as Saccharomyces cerevisiae SG, and has the deposit number of: CCTCC M20221770.
2. The highly active and stress-resistant yeast of claim 1 is applied to anaerobic ethanol fermentation of a synthetic culture medium or a toxic hydrolysate culture medium in a high-temperature environment and a high-sugar environment.
3. The use of the highly active anti-retroyeast according to claim 2, wherein the concentration of fermentation sugar in the synthetic medium and the toxic hydrolysate medium is 20-240g/L.
4. The application of the highly active and stress-resistant yeast as claimed in claim 2 or 3, wherein the toxic hydrolysate is a supernatant containing fermentation sugar and inhibitor obtained by pretreating straw raw materials with dilute acid and performing solid-liquid separation.
5. The use of highly active anti-retrograded yeast according to claim 2 or 3, wherein the temperature of the high temperature environment is 36-45 ℃.
6. The use of highly active and stress-resistant yeast according to claim 4, wherein the high temperature environment temperature is 36-45 ℃.
7. The use of highly active anti-retroyeast according to claim 2 or 3 or 6, wherein the concentration of high sugar environmental sugar is 100-240g/L.
8. Use of highly active antiretroyeast according to claim 2 or 3 or 6, characterised in that it comprises the following steps:
(1) Diluting and coating the high-activity anti-stress yeast preservation solution on a YPD solid culture medium under an aseptic condition, and performing static culture at a constant temperature of 30-45 ℃ for 12-48h to obtain a single colony for liquid activation culture;
(2) Under the aseptic condition, inoculating the single colony in the step (1) into a YPD liquid culture medium, carrying out shake culture at the temperature of 30-45 ℃ and the rpm of 150-200 for 24-48h, and preparing an activated bacterial liquid for anaerobic ethanol fermentation culture;
(3) Under the aseptic condition, inoculating the activated bacterium liquid obtained in the step (2) into a liquid synthetic culture medium or a toxic hydrolysis liquid culture medium according to the inoculation amount of 5% -10%, performing closed anaerobic ethanol fermentation culture at the temperature of 30-45 ℃ and the rpm of 150-200 for 24-72h.
9. The ethanol fermentation application of claim 2, 3 or 6, wherein the synthetic medium or hydrolysate medium, the nutrient components comprise 20-240g/L glucose, 6-30g/L yeast extract, 6-15g/L peptone, 0.8-4g/L ammonium sulfate, 0.8-4g/L potassium dihydrogen phosphate, 0.5-2.5g/L urea, 0.5-1.5g/L sodium glutamate, 0.5-1.0g/L magnesium sulfate heptahydrate, 0.001-0.01g/L manganese sulfate monohydrate, 0.05-0.5g/L zinc sulfate heptahydrate, 0.001-0.01g/L copper sulfate pentahydrate, 0.01-0.1g/L calcium chloride, 0.01-0.1g/L ferric chloride hexahydrate, 0.001-0.01g/L potassium iodide, 0.05-0.15g/L inositol, 50.05-0.15g/L biotin, 0.1-0.5g/L biotin, 0.01-0.01 g/L inositol, 0.05-0.15g/L vitamin B, 50.05-0.15g/L niacin, 0.01-0.05g/L niacin and the balance of natural amino-sulfuric acid solution, and the pH is 0.05-1.05-1 g/L HCl.
10. The ethanol fermentation application of claim 8, wherein the nutrient composition of the synthetic medium or hydrolysate medium comprises 20-240g/L glucose, 6-30g/L yeast extract, 6-15g/L peptone, 0.8-4g/L ammonium sulfate, 0.8-4g/L potassium dihydrogen phosphate, 0.5-2.5g/L urea, 0.5-1.5g/L sodium glutamate, 0.5-1.0g/L magnesium sulfate heptahydrate, 0.001-0.01g/L manganese sulfate monohydrate, 0.05-0.5g/L zinc sulfate heptahydrate, 0.001-0.01g/L copper sulfate pentahydrate, 0.01-0.1g/L calcium chloride, 0.01-0.1g/L iron chloride hexahydrate, 0.001-0.01g/L potassium iodide, 0.05-0.15g/L inositol, 3238 zL vitamin B, 3238.01-0.1 g/L vitamin B, 0.01-0.1g/L pyridoxal, 0.01-0.01 g/L sodium sulfate monohydrate, 0.05-0.5g/L sodium sulfate heptahydrate, 0.05-0.5g/L natural amino acids, 320.05-0.1 g/L HCl and the balance of sulfuric acid.
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