Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • 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
  • C12P7/06—Ethanol, i.e. non-beverage
• • 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
  • C12N1/18—Baker's yeast; Brewer's yeast
  • C12N1/185—Saccharomyces isolates
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • 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
  • C12P7/06—Ethanol, i.e. non-beverage
  • C12P7/08—Ethanol, i.e. non-beverage produced as by-product or from waste or cellulosic material substrate
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
  • C12R2001/00—Microorganisms ; Processes using microorganisms
  • C12R2001/645—Fungi ; Processes using fungi
  • C12R2001/85—Saccharomyces
  • C12R2001/865—Saccharomyces cerevisiae
• • 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

Definitions

• the present invention relates to first-generation ethanol-producing yeast strains, yeasts obtained by culturing these strains, and methods for the industrial production of ethanol from said yeasts. More specifically, the present invention relates to three specific strains that exhibit high ethanol tolerance, lower glycerol production than the most successful strains used today in ethanol production processes, and high ethanol yield. . The strains of the invention also have fast ethanol production kinetics.
• the first-generation bioethanol is produced by fermentation of hexoses (six-carbon sugars) contained in biomasses rich in starch (corn kernels, barley, wheat, cassava, potato tubers, etc.) or sucrose (sugar cane). sugar, sugar beet, sugar sorghum, etc.), while second-generation bioethanol is produced by processing cellulose and hemicellulose contained in agricultural residues such as cereal straws, corn stover, residues forestry, timber, energy crops such as switchgrass or short or very short rotation coppice (poplar for example). Processes for obtaining sugar-rich fermentation media from first-generation processes are relatively simple and well controlled.
• sugar plants such as sugar cane, sugar sorghum, sugar beet
• the plant is crushed or cut into pieces and a sweet juice is obtained directly or after soaking in water.
• the alcoholic fermentation can take place from the raw juice obtained, from concentrated juice or concentrated juices such as molasses obtained after extraction of a fraction of the sugars initially present.
• starch-rich plant fractions such as corn kernels
• the starch must first be hydrolysed to glucose which the yeast can then convert to ethanol.
• the typical hydrolysis process consists of a first phase in which the starch chains are converted into shorter chains by the action of an alpha-amylase followed by a fermentation step called SSF (Simultaneous Saccharification and Fermentation) at the same time.
• SSF Simultaneous Saccharification and Fermentation
• sugar beet and cereals are the main resources used for the production of ethanol of agricultural origin.
• the sugars (glucose, fructose or sucrose) contained in sugar plants (sugar beet, sugar cane) and starch plants (cereals such as wheat or corn) are converted into alcohol by an industrial fermentation process using yeasts.
• the alcohol is then distilled and dehydrated to obtain bioethanol.
• Co-products obtained during the production process (grains and pulps) are intended for animal feed.
• the yeasts used by first generation ethanol producers are mainly specialized yeasts that optimize the profitability of the production process. These yeasts include: Ethanol Red ® (Fermentis ® ), Thermosacc ® (Lallemand ® ), Angel Super Alcohol ® (Angel ® ) and Fali ® (AB Mauri ® ).
• the expected qualities of these yeasts are their ability to rapidly produce high concentrations of ethanol and to exhaust sugars from fermentation ranges of temperature and pH representative of industrial conditions. These qualities are particularly sought after in processes using cereals, maize in particular, which generate hydrolysates with high concentrations of sugars. Producers adjust the sugar content of their fermentation medium to be as high as possible while ensuring that the sugar is as quickly and completely as possible converted into ethanol.
• the producer wants the yeast to convert all of the sugars in the medium into ethanol
• the producer wants the overall conversion efficiency of the sugars consumed to be ethanol to be as high as possible and consequently the least possible co-products such as that glycerol are generated during fermentation.
• the present invention relates to Saccharomyces cerevisiae yeast strains which have improved properties compared to specialized yeast strains commonly used in the production of first generation ethanol.
• the subject of the present invention is the yeast strain Saccharomyces cerevisiae 53-137 which was deposited on July 25, 2013 at the CNCM (National Collection of Microorganism Cultures of the Institut Pasteur, 25 rue du Dondel Roux, 75724 Paris, Cedex 15) under number 1-4791 under the terms of the Budapest Treaty.
• the subject of the present invention is also the yeast strain Saccharomyces cerevisiae 53-005 which was deposited on July 25, 2013 at the CNCM (National Collection of Cultures of Microorganisms of the Pasteur Institute, 25 rue du Do Budapest Roux, 75724 Paris , Cedex 15) under number 1-4790 under the terms of the Budapest Treaty.
• the subject of the present invention is also the yeast strain Saccharomyces cerevisiae 53-214 which was deposited on 25 July 2013 at the CNCM (National Collection of Microorganism Cultures of the Pasteur Institute, 25 rue du Do Budapest Roux, 75724 Paris , Cedex 15) under number 1-4792 under the terms of the Budapest Treaty.
• the present invention also relates to a yeast obtained by culturing a yeast strain Saccharomyces cerevisiae selected from the yeast strain Saccharomyces which was deposited on July 25, 2013 at the CNCM under the number 1-4791, the yeast strain Saccharomyces cerevisiae which was filed on July 25, 2013 at the CNCM under the number 1-4790 and the yeast strain Saccharomyces cerevisiae which was filed on July 25, 2013 at the CNCM under the number 1-4792.
• the present invention also relates to the use of a yeast strain Saccharomyces cerevisiae selected from the yeast strain Saccharomyces which was deposited on July 25, 2013 at the CNCM under the number 1-4791, the yeast strain Saccharomyces cerevisiae which was filed on July 25, 2013 at the CNCM under the number 1-4790 and the yeast strain Saccharomyces cerevisiae which was filed on July 25, 2013 at the CNCM under the number 1-4792 or a yeast obtained by cultivation of the yeast. one of these strains, for the production of first generation ethanol from biomass.
• the present invention also relates to a method for producing first generation ethanol from biomass comprising a fermentation step using a yeast strain Saccharomyces cerevisiae selected from the yeast strain Saccharomyces which was deposited on July 25, 2013 at the CNCM under the number 1-4791, the yeast strain Saccharomyces cerevisiae which was filed on July 25, 2013 at the CNCM under the number 1-4790 and the yeast strain Saccharomyces cerevisiae which was filed on July 25, 2013 at the CNCM under No. 1-4792 or a yeast obtained by culturing one of these strains.
• the biomass is rich in sugar and / or starch and is selected from, or derived in particular from, corn, wheat, barley, rye, sorghum, cassava, triticale, apple. land, sweet potato, sugar cane, sugar beet, sugar sorghum.
• the biomass is selected from or sourced from corn, wheat, barley, cassava, sugar beet, sugar cane.
• the present invention also relates to the manufacture of droughts and droughts with added sulfur from fermentation residues obtained during ethanol production processes.
• the present invention relates to three yeast strains Saccharomyces cerevisiae useful for the production of first generation ethanol.
• the three strains of the invention were obtained by a hybridization and selection program. They result from the hybridization between the yeast strain Saccharomyces cerevisiae deposited at the CNCM on September 4, 2008 under the number 1-4072, a strain belonging to the Applicant, and the yeast clone Saccharomyces cerevisiae deposited at the CNCM on June 26, 2013 under number 1-4782, which also belongs to the Depositor.
• Yeast strain 1-4072 is a strain selected by the Applicant as having the highest tolerance to ethanol among a panel of 21 evaluated strains.
• the yeast clone Saccharomyces cerevisiae 1-4782 was selected by the Applicant as having a high ethanol tolerance, although lower than that of the 1-4072 strain, but as having a lower glycerol production than the 1-4072 strain. .
• the hybrid selection program led to the three strains of the invention.
• each of these strains is an alternative to the most successful specialized strains that are currently used in the industrial production of first-generation ethanol.
• the three strains of the invention have a higher tolerance to ethanol than the reference strain 1-4072 and at the same time produce first generation ethanol with a higher yield than the 1-4072 strain. because of a lower production of glycerol, which is a by-product of the fermentation reaction. This good tolerance is observed whatever the temperature (32 ° C, 35 ° C or 38 ° C), the pH (4.0, 5.0 or 5.5) and the mineral nitrogen supply (150 to 500 ppm) during fermentation, making them particularly suitable for the production of first-generation ethanol where fluctuations in fermentation parameters are common.
• the strains of the invention have the advantage to have ethanol production kinetics that is similar to or slightly less than that of the 1-4072 reference strain.
• the invention also relates to a yeast obtained by culturing one of the strains of the invention.
• the methods for culturing a yeast strain are known in the art, and one skilled in the art knows how to optimize the culture conditions for each strain according to its nature.
• biomass is understood to mean all organic matter of plant origin that can become a source of energy after processing.
• biomass is derived from agricultural and agro-food products and / or co-products.
• the biomass is preferably rich in sucrose or starch, and is selected from, or is derived from, for example, maize, wheat, barley, rye, sorghum, cassava, triticale, potato, sweet potato, sugar cane, sugar beet, sugar sorghum.
• the biomass is selected from, or is derived from corn, wheat, barley, or cassava.
• the methods of producing first generation ethanol from biomass and the use of yeasts in the fermentation step are known in the art.
• the most common industrial process uses physical, chemical and biochemical treatments that ultimately aim to allow fermentation of sugars and produce ethanol.
• Several variants of this process exist and are known to those skilled in the art.
• the yeast strains of the invention and yeasts obtained by culturing these strains can be employed in any first generation ethanol production method.
• the invention is particularly applicable to the production of ethanol as a fuel, but also to the production of ethanol for the food, chemical, pharmaceutical and cosmetic industries.
• FIG. 1 Final concentrations at the end of the fermentation tests carried out on liquefied fermentation media according to Example 1.
• A Concentration of ethanol and
• B concentration of glycerol.
• Media A, B and C are described in Table 1.
• Figure 2 Kinetics of ethanol production by 1-4072 strain (A) and hybrids 1-4790 (B), 1-4791 (C) and 1-4792 (D) under different fermentation conditions.
• Example 1 Selection of Parent Strains to Cross with Strain 1-4072
• a panel of 21 strains or clones was tested in alcoholic fermentation. The tests were carried out under conditions of simultaneous saccharification and fermentation (SSF) with an excess of sugars compared to the ethanol conversion capacities of the strains.
• SSF simultaneous saccharification and fermentation
• synthetic media containing starch dextrins with or without the addition of solubles of the distillation residues were used.
• fermentation media prepared from corn flour, soluble fractions, distillation residues and process water in the proportions given in the table below were used. These different elements were obtained from an industrial ethanol production plant using a first generation process.
• the cornmeal mixture was liquified with Liquozyme TM SCDS (Novozymes) at 85 ° C for 3 hours after adjusting the pH to 5.6.
• Liquozyme TM was 0.8 mL / kg of flour used. Before inoculation, the initial pH was adjusted to 5.0 or 4.5; different concentrations of urea (300, 600 and 1000 ppm nitrogen equivalent) were added as shown in the following table. Spyrizyme Fuel Ultra TM (Novozymes) was added at a dose of 0.6 mL / kg of flour. The fermentation temperature was controlled at 32 ° C. The strains were previously propagated in a rich synthetic medium in 500 ml baffle flasks (micro-aerated propagation). Table 1.
• strain deposited at the CNCM under the number 1-4072 was selected as the strain producing the most ethanol of the strains tested.
• strain 1-4782 was selected as a strain producing large amounts of ethanol, although less than 1-4072, and producing lower amounts of glycerol than 1-4072 strain.
• the objective was therefore to obtain, by hybridization, at least one hybrid having an ethanol tolerance greater than that of the 1-4072 strain, a lower glycerol production than that of the 1-4072 strain, and a kinetics ethanol production at least similar to that of strain 1-4072.
• the loss of mass is an indirect indicator of yeast production of ethanol according to the stoichiometric equation: 1 mol glucose 2 mol C0 2 + 2 mol ethanol which makes it possible to globally connect the mass loss mass of the medium in the form of C0 2 produced and evaporated to the mass of ethanol produced.
• hybrid 1-4790 at temperatures of 35 ° C and 38 ° C, for an ethanol production equivalent to that of the 1-4072 strain, glycerol production is decreased by 15 to 20%. The kinetics of loss of mass, however, is lower than that of strain 1-4072.
• hybrid 1-4791 at temperatures of 35 ° C and 38 ° C, the ethanol production is increased by 3.5% over that of the 1-4072 strain, the kinetics of loss of mass is faster than that of the 1-4072 strain, and the production of glycerol is equivalent to that of the 1-4072 strain.
• Tests were carried out in corn flour and solubles fractions of industrial distillation residues. The tests were carried out at different fermentation temperatures, with different nitrogen additions and at different initial pHs. The characteristics that have been studied are: the maximum ethanol tolerance of the strains, the production of glycerol and the kinetics of ethanol production.
• Mass losses of the fermentation media were measured over time. With stabilization of the mass loss, a sample of the fermentation medium was made and an HPLC assay of ethanol and glycerol concentrations was carried out. The ethanol and glycerol masses produced were calculated from the measured concentration and mass of fermentation must at the time of sampling to perform the assay and initial concentration and mass values. Protocol. Fermentation media were prepared from corn meal, soluble fractions, distillation residues, and process water. In order to better simulate industrial production conditions, these different elements were obtained from industrial ethanol production plants using first-generation processes. The proportions of the three industrial components were: corn flour (36% w / w), fraction of solubles from distillation residues (35% w / w) and process water (29% w / w).
• the mixture was liquefied with Liquozyme TM SCDS (Novozymes) at 85 ° C for 3 hours after adjusting the pH to 5.6.
• the dose of Liquozyme TM has been 0.8 mL / kg of flour used.
• the pH was adjusted to 5 or 4 (according to table), different concentrations of urea (150, 250 and 500 ppm nitrogen equivalent) were added.
• Spyrizyme Fuel Ultra TM Novozymes was added at a dose of 0.6 mL / kg of flour.
• the strains were previously propagated in a rich synthetic medium in 500 ml baffle flasks (micro-aerated propagation).
• a yeast cream was prepared from the propagation medium by centrifugation and resuspended in water from the centrifugation pellet. A dry matter was made on the yeast cream and the fermentation medium was inoculated with the cream so as to have a seeding rate of 0.5 g of dry yeast equivalent / kg of medium.
• Test 1 32 ° C, 500 ppm N, pH5
• Test 3 38 ° C, 500 ppm N, pH5
• strain 1-4072 has the fastest ethanol production kinetics of all strains tested.
• the 1-4791 hybrid has ethanol production kinetics that is approximately similar to that of 1-4072 strain.
• the hybrid 1-4790 exhibits the slowest kinetics with delays of up to 10 hours at pH 5.
• the hybrid 1-4791 exhibits: kinetics of ethanol production identical or similar to that of 1-4072, better tolerance to ethanol (+ 2%), lower production of glycerol (-5%), and a better yield of ethanol (+ 2%); the hybrid 1-4790 has: a better tolerance to ethanol than 1-4072 (+ 3%), a lower production of glycerol (-10%) and a better yield of ethanol (+ 2%) but a kinetics ethanol production slower than that of 1-4072.
• the hybrid 1-4792 exhibits: a better tolerance to ethanol than 1-4072 (+ 3%) except in harsh conditions (38 ° C./ pH 4), a kinetics of ethanol production similar to that of 1-4072, lower glycerol production (-5%) and higher ethanol yield (+ 1%).
• the hybrids selected were multiplied, on a pilot scale, in aerated conditions according to a fed-batch scheme well known to those skilled in the art.
• the yeasts obtained were dried according to the usual techniques. These yeast production tests were conducted without any particular problem being found.
• Ethanol production trials were conducted from instant dry yeasts produced in order to verify that the yeast production process does not alter the performance of the strains produced.
• Fermentation media were prepared from corn meal, soluble fractions, distillation residues, and process water. These different elements were obtained from industrial ethanol production plants. The proportions of the three industrial components were: corn flour (36% w / w), fraction of distillation solubles (35% w / w) and water (29% w / w). The mixture was liquefied with Liquozyme SCDS TM (Novozymes) at 85 ° C for 3 hours after adjusting the pH to 5.6. The dose of Liquozyme TM was 0.8 mL / kg of flour used.
• Dry yeasts produced from hybrids 1-4790, 1-4791 and 1-4792 and Ethanol Red TM commercial yeast were propagated on the liquefied medium diluted in water (70% w / w, 30% w / w). / m).
• the seeding rate was 0.5 g dry yeast / kg medium, the pH was adjusted to 5, the propagation temperature was 32 ° C, urea (500 ppm) and Spirizyme Fuel Ultra TM (0.6 mL / kg flour) were added.
• the propagation medium was transferred to the fermentation medium with 10% mass / mass transfer.
• the fermentations were carried out under the conditions described in Table 8. Table 8.
• the industrial strain 1-4790 makes it possible to improve the ethanol productivity by + 2% with an improved ethanol / sugar yield by reducing the amount of glycerol generated by 10%;
• the industrial strain 1-4792 makes it possible to improve the productivity of ethanol by + 1% with an improved ethanol / sugar yield thanks to the reduction of 5% of the quantity of glycerol generated. Table 9.

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