FR2616445A1 - Process for the production of ethanol by means of microorganism strains having high resistance - Google Patents

Abstract

The fermentation broth constituting the starting material is treated with microorganisms belonging to a homothallic diploid strain of Saccharomyces cerevisiae, obtained by passing through selection cycles, a premeiotic mutagenic treatment intended to introduce genetic variability and successive selections. This strain has the characteristic of resisting high concentrations of ethanol, of not being inhibited at glucose concentrations ranging up to 30 % w/v and of producing ethanol at concentrations ranging up to 15 % w/v. The fermentation medium preferably contains not only glucose up to 30 % w/v but also molasses. Use for increasing ethanol production by fermentation.

Description

The present invention relates to a process for the production of ethanol using high resistance microorganism strains. More particularly, the invention refers to a process for the production of ethanol by fermentation in which a particular strain of
Saccharomyces cerevisiae obtained through several cycles of selection, mutagenesis, genetic segregation, strain that is able to withstand high concentrations of ethanol and not be inhibited by high glucose levels in the fermentation broth in order to provide a product more concentrated in alcohol compared to the methods currently in use. The invention further comprises the novel strain of microorganism developed.

 It is known that ethanol can be produced by fermentation from sugar substrates which are made from yeasts with the production of ethyl alcohol and carbonic acid, as substrates advantageously by-products or agricultural waste which must, in general, be subjected to a preliminary hydrolysis in order to separate the various sweet, cellulosic or starchy substances from the corresponding basic monomers. The by-products currently proposed as starting materials for alcoholic fermentations are predominantly cereal surpluses and wood and cellulose residues which, after proper hydrolysis, the basic monomer sugar is glucose.

 The industrial production of ethanol by fermentation is all the more profitable that is higher the final level of ethanol likely to be reached in a short time in the fermentation medium, and this has always been a problem with difficulty soluble, because saccharomycetes, which are the microorganisms of choice & used in alcoholic fermentation, are not resistant to ethanol concentrations greater than 7 to 8% w / v, thus making any subsequent increase in alcohol content impossible of the final product.

 In addition, even the sugar concentration of the fermentable substrate is subject to important limitations because too high levels are an inhibitory factor for yeast fermentation activity.

 Taking into account that the maximum theoretical yield of alcoholic fermentation from glucose is 48% by weight, to obtain ethanol at 15% w / v (even without regard to the tolerance limits of the latter) it would be necessary to provide a substrate at 30% w / v glucose levels, which concentration generally inhibits the fermentation process.

 Therefore, the typical disadvantages of current processes, with which no more than 15% w / v of glucose in the starting substrate and 7 & 8% w / v of ethanol in the final product are exceeded, are those related to dilution. Excessive fluids involved: need for large volumes of fermentation and storage facilities to achieve good productivity values or, conversely, reduced amount of processed substrates per unit of time and low ethanol productivity. In addition, the higher the flow of ethanol at the outlet is diluted the higher the energy costs for subsequent distillation.

 The object of the present invention is therefore to propose a process for the production of ethanol by fermentation, in which higher sugar concentrations of substrate are used than those normally used, and which makes it possible to obtain a product richer in ethanol. compared to previous possibilities.

 Research has attempted to solve this problem by obtaining highly resistant and highly productive strains of Saccharomyces cerevisiae, and by developing optimal compositions of the fermentation broth.

 The yeast strain which is the subject of the present invention indeed has the characteristic of not being inhibited at glucose concentrations of up to 30% w / v and of being able to produce ethanol up to 15% w / v without suffer mortality.

 The method by which the new strain of Saccharomyces cerevisiae was obtained is described below.

 Ten independent strains of high alcohol wine must have been isolated.

 A first selection was made by removing strains which, after 24 hours, had not produced at least 4% ethanol from 30% glucose.

 Of the remaining 4 strains, a drastic selection was made for ethanol resistance; from this procedure came out the selected strain as being definitive & which was given the acronym MI 831.

 Genetic characterization of strain NI 831 was performed by controlling its positive, sporeforming ability and then dissecting 20 tetrads at the micro-manipulator. Spore survival never exceeded the live ratio: 2 deaths / tetrad indicating that the strain is heterozygous due to the presence of a lethal recessive gene. Analysis of the phenotype of clones derived from these spores has demonstrated that the strain should be considered completely wild in terms of nutritional requirements. Characteristics of fermentation capacity and ethanol resistance exhibit complex mode segregation, indicating that these phenotypes are related to polygenic systems. All clones derived from spores are able to sporify. Spore survival in tetrads from these strains is of type 4 live: O death, which indicates that it is diploids balanced and that MI 831 and all clones derived from the latter are homothalle diploids.

 Among the spore-derived clones of strain MI 831, strain MI 861 was selected which had good fermentation capacity and good ethanol resistance.

 The strain MI 861 has been rendered mutagenic by means of ultraviolet irradiation. After two cycles of mitotic division, 20 independent cultures were transferred into the sporulation medium.

The efficiency of sporulation was reduced by 80% compared to controls. Two tetrads were isolated from each sample and the surviving spores (40% survival) were analyzed for their ability to ferment glucose to 30% (values read after 24 hours). Finally, we chose the strain called MI 861 / 10a which, under the conditions described, produced 14% ethanol p / v.

The strain of Saccharomyces cerevisiae MI 861 / 10a was deposited on May 28, 1987 at the Deutsche Sammlung von
Mikroorganismen of Gottingen, in the Federal Republic of Germany, under deposit number DSM 4128.

 Therefore, the specific object of the present invention is a process for the production of ethanol using strains of high resistance microorganism, characterized in that the fermentation broth constituting the starting material is treated with microorganisms belonging to and a diploid homothalle strain of Saccharomyces cerevisiae, obtained after passing through cycles of selection, a premeiotic mutagenic treatment intended to introduce genetic variability, and successive selections having the characteristic of withstanding high concentrations of ethanol, of not inhibit at glucose concentrations up to 30% w / v and produce ethanol at concentrations up to 15% w / v.

 In particular, this strain is Saccharomyces cerevisiae MI 861 / 10a deposited under the number DSM 4128.

 According to the invention, it is also proposed to use as a fermentation medium a broth containing up to 30% w / v of glucose and, in addition, molasses, which, as is known, is the by-product. sugar extraction from sugar beet or cane. Indeed, it has been considered that molasses contains mineral salts, vitamins and in general substances which constitute impurities to be eliminated by the sugar industry, but which are also nutritive elements that are useful for the development of microorganisms. An addition of molasses thus completes the basic sweet substrate, improving the kinetics of the fermentation reaction and leading to substantial increases in yield at certain concentration levels.

 In particular, it has surprisingly been found that by treating substrates containing glucose up to about 30% and molasses between 7.5 and 10% w / v with the subject microorganism, it is possible to achieve a glucose transformation efficiency equal to 100% of the theoretical value.

 According to a preferred embodiment of the invention, the fermentation broth thus contains glucose in a concentration of about 30% w / v and molasses between 7.5 and 10% w / v.

 Obviously, it is of great interest in the practical application to be able to combine the substrate with a low-cost byproduct such as molasses, thus increasing the yield of production by up to 13% over the normal.

 In practice, the method which is the subject of the invention, according to a preferred embodiment, consists in previously cultivating Saccharomyces cerevisiae MI 861 / 10a in a culture medium, under aerobic conditions, and then inoculating it in a fermentation medium containing about 30% w / v glucose and molasses in a concentration of between 7.5 and 10% w / v, developing fermentation under anaerobic or microaerophilic conditions, temperatures between 25 and 35il, and finally separating the biomass of the resulting liquid product.

 Thus, a final ethanol concentration of 15% w / v can be obtained, which is about twice that obtained using currently known methods.

 As mentioned above, this offers the advantage of being able to process flows, both for the starting substrate and the final product, which are more concentrated, and thus to be able to use installations of smaller volumes with significant reductions in the costs of investment; or with useful volumes of the same importance, it has the advantage of being able to treat higher amounts of substrate and achieve higher ethanol productivities. In addition, the costs of the subsequent purification are reduced because of the lower amount of water to be removed.

 Another object of the present invention is a diploid strain, homothalle of Saccharomyces cerevisiae, obtained after passing through cycles of selection, a premeiotic mutagenic treatment intended to introduce genetic variability, and successive selections, strain which has the characteristic of resisting high concentrations of ethanol, not to be inhibited at glucose concentrations up to 30% w / v and to produce ethanol at concentrations up to 15% w / v. In particular, this strain is Saccharomyces cerevisiae MI 861 / 10a, deposited under the number DSn 4128.

 The present invention will now be described by way of illustration but not limitation, with reference to a specific embodiment.

EXAMPLE
The Saccharomyces cerevisiae strain is inoculated into a sample containing a complete culture medium.
MI 861 / 10a and incubated for 48 hours.

The biomass produced is suspended in 5 ml of physiological solution and this suspension is used to inoculate a liquid culture medium in a ratio of 1: 100. The preferred culture media are
YEPS (0.5% yeast extract, 1% bactopeptone, 2% sucrose) and YEPD (0.5% yeast extract, 1% bactopeptone, 2% glucose).

 The cultures are placed at 30 ° C in an incubator at 240 rpm for 18 hours.

 The biomass produced is then separated from the culture broth by centrifugation and recovered with the fermentation medium so as to obtain a final concentration of 109 cells / ml.

 The slurry is collected for fermentation in a box with a sealing cap and a vent for the generated gas and placed at 28 ° C. under a rotation of 240 rpm.

 Glucose consumption and ethanol production are measured at 2-hour intervals by means of sterile samplings (like all the operations described above) and in such volumes, so as not to deplete, on the whole, the total fermentation volume of more than 2.5%.

 The samples were analyzed for solvents by gas chromatography and for sugars by high-pressure liquid chromatography and enzymatic reaction specific for glucose (Beckman Glucose Analyzer).

 Following the procedure described, several substrates of different composition were fermented to compare the results. Some of the data collected are shown in the following table, in which the high values of ethanol concentration attained by the present invention and the positive influence of the addition of molasses can be clearly seen.

BOARD
Fermentation with the Saccharomyces strain
cerevisiae MI 861 / 10a

<tb> Medium <SEP> Glucose <SEP> 30% <SEP> w / v <SEP> Gluc. <SEP> 30% <SEP> + <SEP> Gluc.30 <SEP>% <SEP> +
<tb><SEP> molasses <SEP> 7.5% <SEP> Mel. <SEP> 10 <SEP>%
<tb><SEP> p / v <SEP> p / v
<tb><SEP> 6h <SEP> 0 <SEP> 24h <SEP> 6h <SEP> 8h <SEP> 24h <SEP> 6h <SEP> 24h
<tb>% <SEP> ethanol
<tb> (w / v) <SEP> 6.25 <SEP> 10 <SEP> 10 <SEP> 7.5 <SEP> 10 <SEP> 13 <SEP> 7.5 <SEP> 15
<tb> Effectiveness <SEP>
<tb> of <SEP> trans
<tb> training
<tb> in <SEP>% <SEP> 1.6 <SEP> 66.6 <SEP> 66.6 <SEP> 50 <SEP> 66.6 <SEP> 86.6 <SEP> 50 <SEP> 100
<Tb>
Fermentation volume: 100 ml
Of course, the invention is not limited to the embodiment that has just been described and can be made to it many modifications without departing from the scope of the invention.

Claims (8)

 A process for the production of ethanol using high-strength microorganism strains, characterized in that the fermentation broth constituting the starting material is treated with microorganisms belonging to a homothalle diploid strain of Saccharomyces cerevisiae obtained by passage through cycles of selection, a premeiotic mutagenic treatment intended to introduce genetic variability and successive selections, this strain having the characteristic to withstand high concentrations of ethanol, not to be inhibited at glucose concentrations up to at 30% w / v and to produce ethanol at concentrations up to 15% w / v.  2. Process according to claim 1, in which the strain Saccharomyces cerevisiae MI 861 / 10a is used and deposited under the name DSM 4128 at the Deutsche Sammlung von Mikroorganismen of Göttingen, Federal Republic of Germany.  3. The process according to one of claims 1 or 2, wherein said fermentation broth contains about 30% w / v glucose and molasses.  4. A process according to claim 3, wherein said molasses is contained in the fermentation broth at concentrations above 2.5 tw / v.  5. The process according to claim 4, wherein said molasses is contained in the fermentation broth at concentrations of between 7.5 and 10%.  6. Process for the production of ethanol using high-resistance microorganism strains, in which the Saccharomyces cerevisiae MI 861 / 10a deposited under No. DSM 4128 is first cultivated on a culture medium in the aerobic state, then inoculating it in a fermentation medium containing about 30% w / v glucose and molasses in concentrations of between 7.5 and 10% w / v when fermenting under anaerobic or microaerophilic conditions. , at temperatures between 25 and 35C, and finally separating the biomass of the resulting liquid product.  7. Strain of Saccharomyces cerevisiae diploid homothalle, obtained by passing through cycles of selection, a premeiotic mutagenic treatment in order to introduce genetic variability and successive selections having the characteristic of resisting high concentrations of ethanol, of not being inhibited at glucose concentrations up to 30% w / v and to produce methanol at concentrations up to 15% w / v.  8. A strain according to claim 7, constituted by Saccharomyces Cerevisiae MI 861 / 10a, deposited under the No. DSM 4128 to the Deutsche Sammlung von Mikroorganismen of Göttingen, Federal Republic of Germany.