WO1994000589A1

WO1994000589A1 - Method for cofermenting glucose and xylose into ethanol using a mixed micro-organism culture

Info

Publication number: WO1994000589A1
Application number: PCT/FR1993/000592
Authority: WO
Inventors: Jean-Philippe Delgenes; Jean-Marie Laplace; Renatto Moletta
Original assignee: Institut National De La Recherche Agronomique
Priority date: 1992-06-24
Filing date: 1993-06-16
Publication date: 1994-01-06
Also published as: FR2692908A1; FR2692908B1

Abstract

A method for producing ethanol by fermenting a culture medium provided with a supply of nutrients and a sugar mixture containing glucose and xylose as the major sugars, and acting as the essential carbon source for fermentation, said fermentation being carried out in the presence both of oxygen and of at least one first yeast for converting glucose into ethanol, as well as at least one second yeast of a different genus from the first, for converting xylose into ethanol, and under conditions suitable for development of the yeasts. The method is characterized in that the first yeast is a respirationally deficient mutant of said first yeast for converting glucose into ethanol.

Description

Process for cofer entation of glucose and xylose in ethanol by mixed culture of microorganisms

The present invention relates to a method for producing ethanol by co-fermentation of glucose and xylose by a pair of microorganisms comprising a microorganism fermenting glucose and a microorganism fermenting xylose. The object of the present invention is to provide a process for biologically converting glucose and xylose into ethanol simultaneously in a single reaction zone.

The biomass from agricultural and agro-food products and / or co-products constitutes a considerable reservoir for the production of molecules of industrial interest. This renewable deposit is in fact estimated at 10 tonnes of coal equivalent per year worldwide. Among these molecules, ethanol occupies a very interesting place. This molecule can on the one hand offer massive and diversified outlets for agriculture and on the other hand constitute a substitute for petroleum products whose market is fragile (price, supply). Ethanol can be used in the energy sector (fuels or additives to fossil fuels as octane donors) and in the food sector. In addition, it can constitute a basic molecule for carbochemistry and can therefore have outlets in plastics, paints, rubbers, fibers, films. Regarding the use of ¹ ethanol in the energy sector, the market is estimated at 12 million hectoliters per year at French level. It should be emphasized that biofuels are favorable to the quality of the environment, by limiting the toxic emissions (carbon monoxide, nitrogen compounds).

The transformation of agricultural carbon into ethanol (especially hexoses) by fermentation can be considered technically controlled. However, a significant limitation remains in terms of production costs, given that the price of the raw material accounts for 40 to 70% of the cost of the final product. It is therefore imperative from an economic point of view to use fermentable carbon at low cost. In this context, the use of sugars derived from lignocelluloses appears favorable to remove this limitation. In addition, among the sources of fermentable carbon, lignocelluloses are well placed for their sugar content compared to the dry matter and the level of availability. The use of lignocelluloses for the production of ethanol can on the one hand relate to the co-products (stalks, straws, bagasse) in the case of production of ethanol from agricultural products such as cereals or cane molasses and on the other hand, exclusively concern certain agricultural products (short rotation coppice). The scheme is dependent on the alcoholic conversion of xylose, a sugar derived from hemicelluloses and representing the second sugar in quantitative terms, after glucose of cellulosic origin, of lignocellulosic materials.

Methods for producing ethanol from a substance containing xylose, comprising fermentation of this substance with yeast have been described, in particular in US Pat. Nos. 4,359,534 and 4,368,268 in which the fermentation uses yeasts belonging to the species Pachysolen tannophilus or Pichia stipitis or Pichia segobrensis or Candida shehatae or yeast mutants of the Candida sp. The description of these processes has sparked renewed interest in the conversion of lignocelluloses to ethanol. Indeed, the conversion of major sugar derived from the hemicellulosic fraction (xylose) in addition to that of the major sugar derived from the cellulosic fraction (glucose) would increase the overall yield of conversion of lignocelluloses to ethanol.

The process of alcoholic conversion of glucose by microorganisms of the genus Saccharomyces or Zymomonas as well as the process of alcoholic conversion of xylose by yeasts described above have been widely studied individually from the kinetic, stoichiometric and physiological points of view. Now, it is a question of developing patterns of production of ethanol by fermentation of lignocelluloses which use both glucose derived from cellulose and xylose derived from hemicelluloses.

Although yeasts fermenting xylose can also convert glucose to ethanol, it is preferable to carry out this conversion with a microorganism with high fermentative potential for glucose of the genus Saccharomyces or Zymomonas, whose alcoholic yields and the productivities from this sugar are higher than those obtained from glucose with yeasts like Pichia stipitis, Candida shehatae or Pachysolen tannophilus. Using specific microorganisms of each of the two sugars, it is possible to design two types of processes for converting glucose and xylose to ethanol: a) Processes of parallel fermentation of glucose and xylose following a separation of these two sugars at the hydrolysis stage (hemicellulosic and cellulosic hydrolysates respectively). b) Simultaneous fermentation processes for glucose and xylose by coculture of two microorganisms, one fermenting glucose, the other xylose (total lignocellulosic hydrolyzate).

Simultaneous fermentation processes for glucose and xylose using a glucose-fermenting microorganism associated with a xylose-fermenting microorganism must take into account the need for oxygen supply to achieve an efficient increased conversion of xylose to ethanol by yeasts such as Pichia stipitis, Candida shehatae or Pachysolen tannophilus. The imperative for these yeasts to maintain a balance between the production and consumption of redox cofactors as well as the metabolic characteristics of the conversion of xylose to ethanol are at the origin of the need for a NAD + generator system such as respiratory chain for rapid fermentation of xylose into ethanol (Prior BA, Kilian SG, Du Preez JC, Process Bioche istry, 24 (1), 21-32, (1989).

Preferably also, efforts will be made to remove or reduce the catabolic repression exerted by glucose on the use of xylose in yeasts such as Pichia stipitis, Candida shehatae and Pachysolen tannophilus when they are cultivated on a glucose / xylose mixture. Glucose and xylose co-fermentation tests with immobilized cells of Pichia stipitis CBS 5573 and Saccharomyces cerevisiae CBS 8066 have been described by Grootjen DRJ Meijlink LKHM, Vleesenbeek R. Van der_.Lans RGJM, Luyben K.Ch.M., Enzyme and Microbial Technology, 13, 530-536 (1991). With these yeast strains co-immobilized in the same reactor and using continuous and aerobic culture conditions, a low conversion rate of xylose is obtained despite the use of a high cell concentration of Pichia stipitis. These authors report that the phenomenon observed is due to the fact that Pichia stipitis is deprived of oxygen, this being consumed by the yeast Saccharomyces cerevisiae which ferments the glucose of the sugar mixture studied. The same phenomenon was observed using these two yeast strains in two reactors in series (see: Grootjen DRJ Jansen ML, Van der Lans RGJ and Luyben K. C :. Λ., Enzyme and Microbial Technology, 13, 828- 833, (1991).

Other tests of co-fermentation of glucose and xylose with Saccharomyces cerevisiae associated with Pachysolen tannophilus cultivated in a discontinuous system and in microaerobiosis have been described by Beck MJ Johnson RD, Baker CS, Applied Biochemistry and Biotechnology, 24/25, 415-424, 1990. In these trials, the authors observe, in the absence of a co-fermentation of these two sugars, a sequential use of glucose and xylose. Thus the tests carried out so far in the laboratory have not allowed the development of an effective process for the alcoholic co-fermentation of glucose and xylose in the same reaction zone, the major obstacle being the unavailability of the oxygen transferred to the culture for the yeast used for the fermentation of xylose into ethanol.

The object of the present invention is to provide a process which makes it possible, by co-cultivating a glucose-fermenting microorganism with a xylose-fermenting microorganism, to obtain effective co-fermentation of glucose and xylose into ethanol.

The microorganisms which can be used in the invention are yeasts belonging to species effectively fermenting glucose into ethanol and for example the species Saccharomyces cerevisiae for the fermentation of glucose and yeasts other than the above capable of converting xylose into ethanol, and by example of the species Pichia stipitis and Candida shehatae for the fermentation of xylose.

The subject of the invention is a process for producing ethanol by fermentation, in the presence of oxygen, of a culture medium supplied with nutrients and with a mixture of sugars containing glucose and xylose as major sugars, constituting the essential source of carbon for fermentation, and in the simultaneous presence of at least one first yeast capable of converting glucose into ethanol and of at least one second yeast not belonging to the same genus as the first yeast, capable of converting xylose to ethanol, under culture conditions allowing the development of said yeasts, characterized in that the first yeast is a respiratory deficient mutant of said first yeast capable of converting glucose into ethanol.

It is preferably carried out with continuous or periodic additions of said mixture of sugars, with additional nutrients if necessary, said additions being carried out only when at least 8% and preferably at least 98% of the glucose previously introduced has already been converted. By "nutrients" is meant the well known elements necessary; to the growth of yeasts, and for example of yeast extract, malt extract and m or more mineral salts, in particular ammonium sulfate. These elements do not constitute the essential source of carbon, this being constituted by the abovementioned sugar intake.

We can operate with dispersed cells. es continuous or semi-continuous (fed-batch) trime described in detail below.

One can also operate in high cell density culture. In this case the yeast cells are retained in the fermenter, and this for example with immobilized cells, with flocculated cells or with cells recycled by a technique using a membrane. The fact of operating at a high glucose conversion rate results in the maintenance of a low value of the glucose concentration in the fermentation medium; this is made possible, despite the continuous or periodic supply of a mixture of glucose and xylose, by the fact that the rate of conversion of glucose by the first yeast is high. It is thus possible to lift or reduce the inhibition exerted by glucose on the xylose conversion yeast. Furthermore, the fact of using a first strain with zero or negligible oxygen requirement makes it available for yeast converting xylose. In what follows, the invention will be described in the case where the first yeast is Saccharomyces cerevisiae or Saccharomyces diastaticus, which constitute the species whose use is preferred in the invention. However, it should be understood that any other yeast with good power to convert glucose into ethanol may be suitable after undergoing treatment to transform it into a respiratory deficient mutant.

As examples of such strains, one can mention:

Saccharomyces eliipsoïdeus Saccharomyces carlberqensis Saccharomyces uvaru Saccharomyces fragilis Schizosaccharomyces po be

Schizosaccharomyces species Kluyvero yces arxianus Zygosaccharo yces rouxii Saccharomyces bayanus. Schwanniomyces occidentalis

The operation is preferably carried out with continuous or periodic supply and withdrawal, so as to maintain a substantially constant reaction volume. Preferably, in continuous operation, a feed rate is maintained in the mixture of sugars such that the determination of glucose by HPLC or high performance liquid chromatography in the effluent of the fermenter gives a zero value, that is to say a concentration below the accuracy limit of the assay, estimated at 0.1 g of glucose / liter. It is thus ensured that at any given moment more than 80% of the glucose has been converted.

We can also operate with a diet continuous or periodic without racking (fed-batch) although this is less advantageous. In this case also, a new addition of the sugar mixture will only be carried out when at least 80% (or at least 98%) of the glucose previously introduced has already been converted.

In practice, if the conversion rates fall below the desired value, the sugar supply rate will be reduced; conversely, this feed rate can be increased if the very high conversion rates allow it.

By respiratory-deficient mutant of a certain yeast is meant a mutant which is essentially incapable of assimilating glycerol or which assimilates it only weakly. Thus, for example, a respiratory deficient mutant of Saccharomyces cerevisiae does not assimilate glycerol or practically does not, unlike conventional strains such as CBS 1200 or NCYC 625.

A simple test to determine whether or not a strain is able to assimilate glycerol consists in culturing the strain in petri dishes on solid medium at 30 ° C. for 48 hours.

The culture on a Petri dish with 20 g / l of glucose serves as a reference culture. On this dish after 48 hours of incubation at 30 ° C the colonies have a diameter of about 2mm. The boxes containing lg / 1 of glucose and those containing lg / 1 of glucose + 20 g / 1 of glycerol allow to reveal the respiratory deficient character. Indeed, glycerol is a substrate whose metabolism is only respiratory. As a result, respiratory impaired mutants are unable to use glycerol for their growth. This translates to the box containing glucose 1 g / 1 + glycerol 20 g / 1 by small colonies of 0.1 mm in diameter which do not differ significantly from the colonies observed on the dish containing only 1 g / 1 of glucose. On the contrary, a non-mutated strain such as

CBS 1200 or NCYC 625, cultured on glucose lg / 1 + glycerol 20 g / 1, gives colonies with a diameter of approximately 2 mm, indicating the use of glycerol.

The mutation of Saccharomyces cerevisiae into a respiratory deficient mutant can be carried out according to the method derived from that described by Sionimski et al. (Biochemical and Biophysical Research Communications, 1968, vol.30, N ° 3, pages 232-239) for example as follows: The cells are cultured in a petri dish on a complete agar medium. A drop of 10 mg / l ethydium bromide is placed in the center of the dish. It is found that the cells located in the center of the box have been killed (no development). Colonies are taken from the edge of the area where the cells have been killed by ethydium bromide. A biomass growth test is then carried out on a differential medium (absence of glycerol assimilation). As yeast converting xylose into ethanol, it is possible to use, for example, a yeast of the genus Pichia or of the genus Candida, preferably chosen from the species Pichia stipitis, Candida shehatae, Pachysolen tannophilus, Candida utilis, Candida tropicalis, Candida tenuis or Pichia segobiensis.

The yeast used to convert xylose to ethanol must have growth compatibility with the strain used to ferment glucose, in particular the respiratory deficient mutants of Saccharomyces cerevisiae, for example those obtained from Saccharomyces cerevisiae CBS 1200 or NCYC 625. This compatibility can be easily determined by a simple preliminary test. All Pichia stipitis strains tested - NRRL Y7124, NRRL Y 11543, NRRL Y 11544, NRRL Y11545 - and all Candida shehatae strains tested - NRRL Y12857, NRRL Y 17024, NRRL Y 12 858, NRRL Y 12856, ATCC 22984 - have this characteristic with respect to at least one of the aforementioned strains of Saccharomyces cerevisiae. The use of all these strains without limitation is therefore envisaged in the process described.

All substrates containing D-glucose and D-xylose as major sugars are suitable in the process described provided that they do not contain any constituent which is a potent inhibitor for the process. Any lignocellulosic material containing cellulose and hemicellulose such as wood, straw, bagasse, corn cobs can be used as raw material to provide the sweet hydrolysates usable in the process to which the additional nutritive elements can be provided. necessary for fermentation and which have been defined above.

The process described involves alcoholic fermentation in aqueous medium of D-glucose and D-xylose. The chemical and physical conditions of the medium must also be such that the viability of the two associated microorganisms is maintained. Fermentation is maintained in the temperature range of 15-40 "C, the range of 26-34 ° C being optimum for the rate of ethanol production. Yeast development takes place in the pH range from 3 to 7, optimal development being observed at pH 5.

As seen above, an important element in the preferred embodiment of the process consists in carrying out, continuously or periodically, additions of the sugar mixture only when it has been verified that 80% (or preferably 98%) at least previously introduced glucose has been converted. It is possible to use a mixture of the glucose and xylose sugars composed for example of 10 to 90% of glucose and 90 to 10% of xylose, by weight, and preferably of 50 to 80% of glucose and 50 to 20% of xylose, weight.

If the total concentration of this mixture of sugars in the aqueous sweetened feed solution is between, for example, 5 g / l and the saturation, and preferably between 10 and 150 g / 1, a dilution rate D will advantageously be adopted. , namely a ratio of the hourly volume flow rate of the feed solution to the reaction volume of 0.0001 to 2 h ^-1 and preferably from 0.001 to 1 h ^-1 .

It is understood that the other nutritive elements can be provided as a mixture with the sugars or separately.

The above values are however only given as an indication, since a simple dosage of glucose in the effluent from the fermenter will make it possible to determine the conversion rate of glucose and therefore to know whether it satisfies the above requirements. The continuous culture of the species defined above is done in the presence of oxygen in a culture medium containing the conventional nutrients well known to biochemists and which it is therefore unnecessary to indicate in detail here. This environment contains, among other things, essential mineral salts, vitamins and trace elements.

The aeration rate must be sufficient to allow the fermentation of the sugars, and in particular the xylose, but not excessive, which would reduce the ethanol yield as a result of increased cell production and oxidation of the ethanol produced. The optimum value for air flow can be determined by simple tests. Expressed as the oxygen transfer rate, it is, for example from 0.0001 to 500 and preferably 0.001 to 100 millimoles of oxygen per liter of fermentation medium and per hour. To measure it, it suffices to differentiate between the oxygen introduced into the fermenter and the oxygen discharged from the latter, therefore to make the oxygen balance, and to report the result to the volume unit of fermentation medium. and by the hour unit.

The following examples are intended to more fully describe the process of the invention but should not be considered as limiting the scope of the invention defined by the claims. General experimental procedure;

By treatment with ethydium bromide, a respiratory deficient mutant of each of the following two strains was first prepared: Saccharomyces cerevisiae: strains CBS 1200 (described by Hoppe et al., Biotechnology. Letters, 1982, vol.4 (1 ) page 39 and Maiorella et al., Biotechnology and Bioengineering, 1984, 26, page 1155, obtained from the Central Bureau Voor Schimmelculture, Delft; strain NCYC 525 (described by Spencer et al., Current Geπetics, 1983, 7, page 159 and Kleinman MJ et al., Biochem J, 1988, 249, page 163 obtained from the National Council Yeast Collection, Norwich, Great Britain.

Slanted cultures on Pichia stipitis agar; strains NRRL Y7124 (described by Dellweg et Coll, Biotechnology Letters, 1934, 6, page 395, and Delgenes et Coll., Biotechnology Bioengineering, 1989, 34, page 398), NRRL Y11543 (described by Dellweg et Coll., Biotechnology Letters, 1984, 6, page 695), NRRL Y11544 (described by Toivola et Coll., Applied Environ ental Microbiology, 1984, 47, page 1221), NRRL Y 11545 (described by Toivola et Coll., Applied Environmental Microbioloby, 1984, 47, page 1221) and Candida shehatae strains NRRL Y 12857 (described by Slininçer et Coll., Biotechnology Letters, 1985, 7, page 431), NRRL Y 12858 (described by Bruinenberg et Coll., Applied Microbiology Biotechnology, 1984, 19, page 256), NRRL Y 12856 (described by Slininger et al., Biotechnology Letters, 1985, 7, page 431), NRRL Y 17024 (described by Kurtz an, Antonie Van Leeuwenhoek, 1990, 57, page 215) were obtained from Northern Regional Research Center, Peoria Illinois. An inclined culture of Candida shehatae ATCC 22984 (described by Dupreez JC et Coll., Biotechnology Letters, 1985, 7, page 241 and Laplace JM et Coll., Applied Microbiology and Biotechnology, 1991, 36, page 158) was obtained from the American Type Culture Collection Rockville, Midland. The strains were maintained on slants of glucose at 30 ^° C. The inciinated culture medium contained 10 g / l of glucose, 5 g / l of pancreatic peptone, 3 g / l of yeast extract, 3 g / l of malt extract and 20 ç / 1 of agar. The liquid media for the propagation of yeasts contained 5 g / l of ammonium sulphate, 3 g / l of malt extract, 3 g / l of yeast extract for the time necessary for significant consumption of the substrate. The carbon source consisted of glucose alone for Saccharomyces and xylosa alone for yeasts of the genus Pichia and Candida at a concentration of 50 ç / 1. The composition of the fermentation medium differed only in the carbon source, which consisted of a mixture of lucose / xylose at respective concentrations of 35 g / 1 and 15 g / 1, proportions which simulate a total hydrolyzate of lignocelluloses. For the natural environment, the carbon source is provided by a total hydrolyzate of poplar wood obtained enzymatically and diluted so as to have an overall sugar concentration of 50 g / 1.

To carry out the fermentation, the liquid medium containing 5 g / l of ammonium sulphate, 3 g / l of malt extract and 3 g / l of yeast extract was introduced into an aerated fermenter and the mixture was fed with sugar mixture (50 g / 1 solution of the sugar mixture additionally providing 5 ç / 1 of ammonium sulphate, 3 ç / 1 of malt extract and 3 ç / 1 of yeast extract) at a rate positive but limited so as to permanently have a glucose conversion in the fermenter greater than 90% and preferably close to 100%, this in the case of continuous operation.

Under these conditions we were able to obtain a xylose conversion greater than 40% and even up to 100%.

The 50 g / l solution of the mixture of sugars was produced in a liquid medium of the same composition as that initially introduced into the fermenter (ammonium sulfate -5- malt and yeast extracts), the whole bringing all the elements necessary for the growth of yeasts, in non-limiting quantities. Unless otherwise indicated, one performed at 30 ^° C, pK = regulated 5- and an aeration rate of 0.005 v / v / min.

The ethanol produced was measured by gas chromatography. D-glucose and D-xylose were quantified by HPLC (high performance liquid chromatography). The total cell development expressed in g / 1 (dry weight) was measured by gravimetry. The respective cell proportions of the yeast used to ferment glucose and the yeast used to ferment xylose, expressed as a percentage of the total number of cells present in the fermenter, were determined in Petri dishes on the basis of the inability of the respiratory deficient mutant to use glycerol as a carbon source for its growth.

In the studies described below, the different values given in the tables were calculated as follows from the dosages carried out on the samples:

* Alcoholic yield, Y _p / _S (g of ethanol produced per g of sugar consumed) Y _p / _S = where E _j = ethanol concentration at the outlet

S _Q -Sf S _Q = sugar concentration at the inlet and Sf = sugar concentration at the outlet. feed rate

* Dilution rate, D =, in h ^-1 reaction volume * Volumetric productivity in ethanol, Qp (g of ethanol produced per liter of reaction volume per hour).

Qp = E _f .D where D is the dilution rate as defined above. In the examples which follow, respiratory deficient mutants of Saccharomyces cerevisiae CBS liOO N "1-1216 and of Saccharomyces diastaticus (cerevisiae) NCYC 625 N ° 1-1217 which were prepared from the mother strains were used by the method of ethydium bromide described above. It was then found that these mutants did not assimilate glycerol by proceeding according to the test described above.

Co-fermentation of the model sugar mixture (35 g / 1 of glucose + 15 g / 1 of xylose) by the respiratory deficient mutant of Saccharomyces cerevisiae CBS 1200 and Candida shehatae ATCC 22984. The experiment was carried out in accordance with the procedure exp * ^~ general mental with an aeration flow of 0, vvm and a stirring speed of 800 revolutions / min, corresponding to an oxygen transfer speed of 3.9 mmol / l / h. The fermentation parameters calculated during the stationary state are reported in table 1,

TABLE 1

As shown in Table 1, the process of the invention made it possible to obtain the simultaneous fermentation of glucose and xylose into ethanol in the same reactor by the pair of yeasts comprising the respiratory deficient mutant of Saccharomyces cerevisiae associated with Candida shehatae. EXAMPLE 2:

Co-fermentation of the same mixture of sugars as in Example 1 by the respiratory deficient mutant of Saccharomyces cerevisiae NCYC 625 and Pichia stipitis NRRL Y 7124 with a reactor of the gazosiphon type. We operated at high cell density using flocculation as a cell retention tool.

The experiment was carried out in accordance with the general experimental procedure. Three dilution rates were tested. For each dilution rate, the fermentation parameters were calculated during the steady state. They are presented in Table 2. TABLE 2

For the three dilution rates tested. all of the glucose and xylose is consumed. The alcoholic yield of 0.41 g / g corresponds to 80% of the theoretical yield. The best alcoholic yield obtained with the mutant respiratory deficient couple of Saccharomyces cerevisiae NCYC 625 and Pichia stipitis NRRL Y 7124 compared to the couple tested in Example 1 relates to a lower production of xylitol by Pichia stipitis compared to Candida shehatae. EXAMPLE 3:

Co-fermentation of the same mixture of sugars as in Example 1 by the respiratory deficient mutant of Saccharomyces cerevisiae CBS 1200 and Pichia stipitis NRRL Y 11545. The experiment was carried out in accordance with the general experimental procedure. The parameters gathered in table 3 are calculated during the steady state. The flow aeration was 0.005 v / v / m with stirring of 800 revolutions / n or an oxygen transfer speed of 1.7 mmol / 1 / h.

TABLE 3

EXAMPLE 4:

Co-fermentation of the model sugar mixture by the respiratory deficient mutant of Saccharomyces cerevisiae CBS 1200 and Candida shehatae ATCC 22984 according to the fed-batch process, that is to say without racking and with successive contributions of sugars (5 g at a time). Each intake was made when at least 90% of the glucose from the previous intake had disappeared. These contributions were made in the form of a 150 g / l solution (112.5 g / l of glucose and 37.5 g / l of xylose) additionally providing the ammonium sulphate and the yeast and malt at the concentrations indicated above (respectively 5g / l, 3 g / 1 and 3g / l). The reactor was aerated at a rate of 0.01 vvm. Agitation was done at 800 rpm. The results obtained are shown in Table 4. TABLE 4

Experimental results (examples 1, 2 and

3) have shown that the implementation of continuous culture conditions makes it possible to lift the glucose effect and by extension leads to the co-fermentation of glucose and xylose by the pair of yeasts retained. The fed-batch process can also create this type of condition, as shown by the results presented in Table 4 with the respiratory deficient mutant of Saccharomyces cerevisiae CBS 1200 and

Candida shehatae ATCC 22984.

EXAMPLE 5:

Co-fermentation of a total poplar wood hydrolyzate by the respiratory deficient mutant of

Saccharomyces cerevisiae NCYC 625 and Pichia stipitis NRRL Y7124. The total hydrolyzate had the following composition:

Cellobiose 11 g / kg

Glucose 329 g / kg

Xylose 72 g / kg Total sugars 412 g / kg

This hydrolyzate was diluted with water to obtain an initial sugar concentration of 50 g / l (41 g / 1 of glucose and 9 g / 1 of xylose).

The experiment was carried out in accordance with the general experimental procedure, with a gazosiphon reactor. Table 5 gives the results obtained:

TABLE 5

It should be noted that in example 5, a crude hydrolyzate was used and that higher yields of ethanol can be obtained if the crude hydrolyzate is subjected to a preliminary purification treatment, for example by exchange resin. ions or by liquid - liquid extraction with a solvent.

It should also be noted that, in the previous tests, one can replace Candida shehatae ATCC 22984 by Candida shehatae CSIR 599, replace Pichia stipitis NRRL 7124 by Pichia stipitis ATCC 58376 or CBS 577 and Pichia stipitis NRRL 11545 by Pichia stipitis ATCC 58785 or CBS 6054 in order to obtain substantially equivalent results. Likewise, respiratory mutants can be replaced. Saccharomyces cerevisiae CBS 1200 and NCYC 625 by respiratory deficient mutants of ATCC 4126 and IFO 1958.

The respiratory deficient mutant obtained from Saccharomyces cerevisiae CBS 1200 and bearing the internal reference CD 2304 has been deposited in the Collections of the Institut Pasteur under the number 1-1216.

The respiratory deficient mutant obtained from Saccharomyces diastaticus (= Saccharomyces cerevisiae) NCY 625 and bearing the internal reference

TN 1809 has been deposited in the Collections of the Institut Pasteur under the number 1-1217.

These new strains of mutants of Saccharomyces cerevisiae, respectively listed in the Collection of Cultures CNCM of the Institut Pasteur in Paris under n ° 1-1216 and 1-1217, have allowed to obtain good results when they are used. work in the process of the invention.

Claims

1. Process for the production of ethanol by fermentation in the presence of oxygen of a culture medium supplied with nutrients and a mixture of sugars containing glucose and xylose as major sugars, constituting the essential source of carbon for fermentation, and in the simultaneous presence of at least a first yeast capable of converting glucose into ethanol and of at least a second yeast not belonging to the same genus as the first yeast, capable of converting xylose into ethanol, in culture conditions allowing the development of said yeasts, characterized in that the first yeast is a respiratory deficient mutant of said first yeast capable of converting glucose into ethanol.

2. Method according to claim 1, in which one operates with continuous or periodic additions of said mixture of sugars, with if necessary additional nutritive elements, said additions being carried out only when at least 80% of the glucose previously introduced has already been converted.

3. The method of claim 2, wherein said additions are only made when at least 98% of the glucose previously introduced has already been converted.

4. Method according to any one of claims 1 to 3, wherein further is withdrawn continuously or periodically a portion of the mixture present in the fermentation zone so as to maintain a volume of fermentation medium substantially constant. 5. Method according to one of claims 1 to 3 wherein it is fed continuously or periodically without withdrawing fermentation medium.

6. Method ^ according to any one of claims 1 to 5, wherein the respiratory deficient mutant capable of converting glucose to ethanol is a respiratory deficient mutant of Saccharomyces cerevisiae or Saccharomyces diastaticus and the yeast capable of converting xylose to ethanol is of the genus Pichia or of the genus Candida.

7. Method according to any one of claims 1 to 5, in which the respiratory deficient mutant capable of converting glucose into ethanol is a respiratory mutant of one of the strains:

Saccharomyces ellipsoïdeus Saccharomyces carlberqensis Saccharomyces uvarum Saccharomyces fragilis Schizosaccharomyces pombe

Schizosaccharomyces species Kluyveromyces marxianus Zygosaccharomyces rouxii Saccharomyces bayanus Schwanniomyces occidentalis and the yeast capable of converting xylose to ethanol is of the genus Pichia of the genus Candida or of the genus Pachysolen.

8. The method of claim 6 or claim 7, wherein the yeast capable of converting xylose to ethanol is of the species Pichia stipitis or Candida shehatae, Pachysolen tannophilus, Candida utilis, Cndida tropicalis, Candida tenuis, or Pichia segobiensis.

9. Method according to any one of claims 1 to 8, in which the yeast converting xylose into ethanol is chosen from the Pichia stipitis strains NRRL Y 7124, NRRL Y 11 543, NRRL Y 11544, NRRL Y 11545, ATCC 58376, CBS 577, ATCC 58785 or CBS 6054 and the Candida shehatae strains NRRL Y 12857, NRRL Y 17024, NRRL Y 12858, NRRRL Y 12856, ATCC 22984 and CSIR 599. 10. Method according to any one of claims 1 to 9, in which mixture of sugars contains from 10 to 90% by weight of glucose and 90 to 10% by weight of xylose, and preferably from 50 to 80% by weight of glucose and 50 to 20% by weight of xylose.

11. Method according to any one of claims 1 to 10, in which the mixture of sugars is supplied in the form of a solution of said sugars with a concentration of 5 g / 1 until saturation and preferably 10 to 150 g / 1 in a mixture of sugars.

12. Method according to any one of claims 1 to 11, in which the dilution rate, expressed as a ratio of the hourly volume flow rate of feed to the reaction volume, is from 0.0001 to 2 h ^-1 and preferably from 0.001 at l ^~ .

13. Method according to any one of claims 1 to 12, in which the air flow supplying the oxygen necessary for the yeast converting the xylose expressed as the oxygen transfer rate, is from 0.0001 to 500 and from preferably 0.001 to 100 millimoles of oxygen per liter of fermentation medium per hour. i4. Process according to any one of Claims 1 to 13, in which the operation is carried out with yeasts dispersed in the culture medium or with cultures with high cell density.

15. Strain (s) of mutants of Saccharomyces cerevisiae respectively deposited under the numbers I-1215 and 1-1217 in the CNCM Collection.