WO2010103530A1

WO2010103530A1 - Method for producing ethanol by c-5 sugar fermenting saccharomyces strain

Info

Publication number: WO2010103530A1
Application number: PCT/IN2009/000491
Authority: WO
Inventors: Samir S. Somaiya; Sangeeta Srivastava; Martin Annette
Original assignee: Godavari Biorefineries Ltd.
Priority date: 2009-03-09
Filing date: 2009-09-08
Publication date: 2010-09-16

Abstract

The present invention relates to a Saccharomyces cerevisiae MTCC 5452 strain capable of fermenting C-5 sugar which is not genetically modified strain. The present invention also provides a fermentative method for producing ethanol with the said strain, comprising incubating culture of Saccharomyces cerevisiae MTCC 5452 with a fermentation medium comprising at least C-5 sugar carbon source under favourable pH and at temperature and fermentation conditions for a sufficient period to obtain ethanol with higher productivity.

Description

METHOD FOR PRODUCING ETHANOL BY C-5 SUGAR FERMENTING SACCHAROMYCES STRAIN.

FIELD OF THE INVENTION

The present invention relates to a method for producing ethanol by fermenting C-5 sugars with Saccharomyces cerevisiae strain. More particularly the present invention relates to a Saccharomyces cerevisiae MTCC 5452 strain capable of fermenting C-5 sugar and a fermentative method of producing ethanol with the help of the said strain.

BACKGROUND OF THE INVENTION

Cellulose, hemicellulose are the most abundant renewable organic resources. These renewable organic resources can be used for the production of fuels like ethanol by the fermentation process using the easily available microorganisms like yeast.

The conversion of cellulose by yeast for the production of fuel ethanol can be carried out very efficiently, as glucose is the main constituent of the cellulose hydrolysate and is utilized very easily by the Yeast. However, conversion of hemicellulose for the production of fuel is problematic, as hemicellulose is mainly composed of pentose sugars, wherein xylose is present in the large amount in the hydrolysate. The conventionally used yeast strains cannot utilize substrates with five carbon atoms mainly xylose (Barnett et al. 'Yeasts Characteristics and Identification' 2^nd edition (1990), Cambridge University Press). Thus, fermentation of pentose sugars is problematic. Most commonly used yeast Saccharomyces cerevisiae ferments the hexose sugars glucose, galactose and mannose to provide ethanol, but is unable to ferment the pentose sugars. There have been various approaches that have been adopted to ferment the C-5 sugar, particularly xylose for producing ethanol.

One of the approaches suggests use of artificial adapted microorganisms. US Patent 5620877 (1997) discloses fermentation of pentose and hexose sugars using respiratory deficient strain of Saccharomyces cerevisiae that had been previously grown on 5% xylose media for 1-2 weeks before being used on the mixed sugars. Fermentation method is slow, and productivity is low due to which the process may not be commercially viable. There have also been attempts to use non-recombinantely modified strains of S. cerevisiae for fermentation of xylose. US4511656A (1985) discloses mutant strain of S. cerevisiae to ferment xylose. However, it showed very low ethanol productivity. WO2005121337A1 describes a non-recombinant method for producing genetically modified Saccharomyces strains by growing cells on medium with xylose as a sole carbon source for two months and growing of the new populations of heterogeneous Saccharomyces cells in liquid culture in xylose minimal medium for approximately 50 generations. Thus it is extremely lengthy and cumbersome.

Another commonly used approach is of two stage process utilizing separate microorganisms. For example US20030199049 (2003), discloses use of adapted yeast strain Pichia stipitis in the first-stage for xylose fermentation and Saccharomyces cerevisiae mutant strain for fermentation of glucose in the second-stage. Further such approach is suggested in two separate patent applications filed by the same applicant. The first stage of the process is disclosed in Brazilian Patent Application Pl 0505299-8 wherein xylose is fermented by Pichia stipitis to obtain ethanol. The second application, WO 2008065433 discloses use of non genetically- modified Saccharomyces cerevisiae yeast for fermentation of hexose. Yet another approach of is use of mix culture for example US20070231869A1 (2007) employs S. cerevisiae along with mixture of fungi, wherein S. cerevisiae is used for fermentation of hexoses whereas mixture of fungi used are capable of metabolizing 5- carbon or 6-carbon sugars. The fermentation process with such mixture however requires very long hours and productivity is very poor.

Recently there have been attempts to use recombinant Saccharomyces cerevisiae for example S. cerevisiae has been genetically engineered to metabolize and ferment xylose the genes for XR and XDH from the xylose fermenting yeast Pichia stipitis have been expressed in S. cerevisiae (European Patent to C. Hollenberg. 1991; Hallborn et al., "Recombinant yeasts containing the DNA sequences coding for xylose reductase and " xylitol dehydrogenase enzymes", WO91015588; Kbtter and Ciriacy, "Xylose fermentation by Saccharomyces cerevisiae", Appl. Microbiol. Biotechnol. 38:776-783, 1993). These transformants metabolize xylose but do not ferment the pentose sugar to ethanol. In another method proposed Xylose isomerase (Xl) from the thermophilic bacterium Thermus thermophilus was expressed in S. cerevisiae, and the recombinant strain fermented xylose to ethanol (Walfridsson et aJ., "Ethanolic fermentation of xylose with Saccharomyces cerevisiae harboring the Thermus thermophilus xylA gene which expresses an active xylose (glucose) isomerase", Appl. Environ. Microbiol. 62:4648-4651, 1996). The low level of ethanol produced was assumed to be due to the fact that the temperature optimum of the enzyme is 85 degree Celsius, whereas the optimum temperature for yeast fermentation is 30 degree Celsius. WO2005118828A1 uses genetically modified organisms Saccharomyces cerevisiae (GMO), such as VTT strain B- 03339 for fermentation of pentose sugars. However, productivity of such method is very low. Besides the problem of low productivity or complexities involved in modifying characteristics and/or introducing genes from different species by recombinant methods, the use of recombinant DNA techniques reduces the opportunities and desirability to use yeasts in human foods and other such applications. If recombinant techniques are used to develop Saccharomyces cerevisiae, they lose the GRAS status. There are also social, economical, environmental, political or other barriers for use of strains that are derived through recombinant- DNA techniques. It is therefore not desirable or suitable to use strains of Saccharomyces cerevisiae that contain genes from other genera or species, or that are produced using recombinant DNA methods.

The above prior art approaches for the fermentation of C-5 sugars for producing ethanol the processes are characterized by, lengthy and cumbersome processes and poor yield. Hence, there is a need to develop a bioprocess for producing ethanol using such a microbe that can provide high yield utilizing resources which are otherwise difficult to be utilized and provide a low cost and efficient process.

SUMMARY OF INVENTION:

Accordingly, the present invention makes available a method for producing ethanol by fermenting atleast C-5 sugars with Saccharomyces cerevisiae strain MTCC 5452.

The present invention in one of the aspect provides a method for producing ethanol with higher productivity by fermenting xylose with C-5 fermenting Saccharomyces cerevisiae strain MTCC 5452.

In another aspect the present invention also provides a method for producing ethanol by further fermenting C-6 sugars with Saccharomyces cerevisiae strain MTCC 5452. The present invention also makes available a Saccharomyces cerevisiae MTCC 5452 strain which is capable of fermenting atleast C-5 sugar to produce ethanol.

In still another aspect the present invention makes available a Saccharomyces cerevisiae MTCC 5452 strain which is capable of further fermenting C-6 sugar to produce ethanol.

In preferable aspects the present invention provides a Saccharomyces cerevisiae MTCC 5452 strain which is not modified by recombinant techniques.

In another aspect the present invention provides a starter culture comprising of cells of Saccharomyces cerevisiae MTCC 5452.

Further aspects of the present invention as defined in the claims, incorporated herein by reference, and advantages associated with the invention, will be evident to a skilled person from the description and examples.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will be described in conjunction with reference to the following drawings, in which:

Figure 1 is a graph illustrating ethanol productivity by Saccharomyces cerevisiae strain MTCC 5452 as per example 4.

Figure 2 is a graph illustrating ethanol productivity by Saccharomyces cerevisiae strain MTCC 5452 as per example 5.

Figure 3 is a graph illustrating ethanol productivity by Saccharomyces cerevisiae strain MTCC 5452 as per example 6. DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a method for producing ethanol by fermenting medium comprising at least C-5 sugar with Saccharomyces cerevisiae strain MTCC 5452. The present invention in another aspect also provides a novel microorganism Saccharomyces cerevisiae MTCC 5452 having capability of fermenting at least C-5 sugar and providing higher productivity of ethanol.

As used herein the term "C-5 sugar" refers to 5 carbon containing sugar, for example selected from group consisting of but not limited to xylose, arabinose, ribose and any monosaccharide comprising 5 carbon atom that can be fermented by Saccharomyces cerevisiae MTCC 5452 strain of the present invention.

The term "C-6 sugar" refers to 6 carbon containing sugar, for example selected from group consisting of but not limited to glucose, fructose, mannose, galactose, rhamnose, and any monosaccharide comprising 5 carbon atom that can be fermented by Saccharomyces cerevisiae MTCC 5452 strain of the present invention.

In one embodiment the invention provides a novel strain of Saccharomyces cerevisiae MTCC 5452 having capability of fermenting C-5 sugar and/or C-6 sugar thereby able to produce ethanol with higher productivity.

In the preferred embodiment of the present invention the novel strain of Saccharomyces cerevisiae is isolated from nature, more preferably from soil environment, by collecting the soil samples from the vicinity of Somaiya Organo Chemicals distillery, located at, Sakharwadi, Ahmednagar, Maharashtra, India. The novel isolated microorganism has been identified to be Saccharomyces cerevisiae based on the following taxonomical characteristics: Gram-positive rods of dimensions 32 to 35 nanometer;

Formation of ellipsoid or cylindrical spores; immobile;

Aerobic to facultative anaerobic;

Growth at 25 degree Celsius to 37 degree Celsius, optimum temperature at 25 degree Celsius to 30 degree Celsius;

Growth at pH value of 5.5 to 6.0, optimum pH range between5 To 6.

The above characteristic confirms that the novel isolated microorganism is Saccharomyces cerevisiae. The microorganism however has a different biochemical profile as compared to the known Saccharomyces cerevisiae as provided in Table 1 herein below:

Table 1

From the above characteristics it is confirmed that the novel isolated Saccharomyces cerevisiae of the present invention has a capability of fermenting xylose sugar. Since the isolated Saccharomyces cerevisiae is not identical and differs in this regard from any known naturally occurring Saccharomyces cerevisiae but has identical taxonomic characteristic, it was confirmed that it is a novel strain which belongs to species Saccharomyces cerevisiae. The advantageous feature of the present invention Saccharomyces cerevisiae strain includes its capability to ferment five carbon sugars for example xylose.

The novel isolated strain Saccharomyces cerevisiae has been deposited with Microbial Type Culture Collection and Gene Bank (MTCC), at Institute of Microbial Technology (IMTECH), Sector39-A, Chandigarh, India, on 4th December, 2008 under the accession number MTCC 5452. Included within the scope of the present invention are the novel isolated strain Saccharomyces cerevisiae MTCC 5452 and also subclones, progeny, cultures and subcultures of this isolate. In the most preferred embodiment of the present invention the novel isolated strain of Saccharomyces cerevisiae is not genetically modified by recombinant DNA techniques. Although, Saccharomyces cerevisiae MTCC 5452 strain is useful, particularly in its isolated form, it may be possible to subject it to recombinant DNA techniques resulting in the generation of new strains having improved properties. For example, the improved properties may include the higher fermentation production rates, various tolerance capabilities, production of fewer byproducts, or optimized for specific fermentation parameters including temperature, pH, carbon or other nutrient requirements. Such strains modified by recombinant DNA techniques are included within the scope of the present invention.

The present invention in one embodiment provides a method for producing ethanol by fermenting medium comprising at least C-5 sugar with Saccharomyces cerevisiae strain MTCC 5452.

An exemplary preferred embodiment of the present invention method for producing ethanol by fermenting C-5 sugars with Saccharomyces cerevisiae MTCC 5452 comprises; incubating culture of Saccharomyces cerevisiae MTCC 5452 with a medium comprising atleast C-5 sugar carbon source under favourable pH and at temperature and fermentation conditions for a sufficient period to obtain ethanol; recovering ethanol from the fermentation broth by appropriate method.

In the preferred embodiments of the present invention Saccharomyces cerevisiae MTCC 5452 is used alone to ferment substrate in a single stage to produce ethanol. However, combination of Saccharomyces cerevisiae MTCC 5452 with other microorganism to produce ethanol either in a single or multiple stage may be within the scope of the present invention.

In the preferred embodiments C-5 sugar is used as a carbon source. The present invention strain Saccharomyces cerevisiae MTCC 5452 has capability of utilizing C-5 sugar as sole carbon source. The most preferred C-5 sugar used as a carbon source is xylose, arabinose, ribose or any other C-5 sugar which may be fermented by the novel strain of the present invention. In alternate embodiments, the medium may comprise of C-6 sugars as a carbon source. The C-6 sugars may be selected from but not limiting to glucose, fructose, galactose, rhamnose, mannose, and other monosaccharides which may be fermented by the novel strain of the present invention.

In still other embodiments, the medium may comprise combination of C-5 as well as C-6 sugars as a carbon source.

In one of the embodiments, as source of C-5 sugar, hemicellulose hydrolysate may be used. In another embodiment, as a source of C-6 sugar cellulose hydrolysate may be used.

In preferred embodiments of the present invention the hemicellulose hydrolysate and/or cellulose hydrolysate may be obtained from lignocellulose containing material, by subjecting the lignocellulose containing material to autohydrolysis. Autohydrolysis may be selected from atleast one treatment including hydrothermal treatment or steam explosion. Hydrothermal treatment may be carried out as may be known to those skilled in the art that is by treating the lignocellulose containing material with hot water preferably compressed water. Similarly, the steam explosion may be carried out as known to the person skilled in the art. Autohydrolysis may be carried out in neutral, acidic or alkaline condition, for a sufficient time so as to degrade the lignocellulose containing material and liberate the hemicellulose and/or cellulose fraction and partially or completely hydrolyze these fractions into monosaccharides including C-5 and C-6 sugars.

In alternate embodiments, the hemicellulose hydrolysate may be obtained by subjecting the hemicellulose fraction of the lignocellulose containing material to atleast one of the processes selected from acid hydrolysis or treatment with enzyme having hemicellulolytic activity. For example, hemicellulase can be added to degrade hemicellulose and obtain C-5 sugar containing hemicellulose hydrolysate.

The cellulose hydrolysate may be obtained by subjecting the cellulose fraction of lignocellulose containing material to at least one of the process selected from acid hydrolysis or treatment with enzyme having cellulolytic activity. For example, cellulase can be added to degrade cellulose and obtain C-6 sugar containing cellulose hydrolysate.

The hemicellulose and/or cellulose hydrolysate obtained by any of the method as herein above described may be used for fermentation having the sugar concentration of at least 8%, preferably in the range of 8-12%.ln some of the embodiments, fermentation of carbon source comprising C-5 sugar and/or C-6 sugar may be carried out at the same time of hydrolysis of lignocellulose containing material or hemicellulose fraction and/or cellulose fraction.

The lignocellulose containing material may be selected from variety of materials. Relevant source of lignocellulose containing material includes wooden biomass from any species of softwood or hardwood trees. Source of wooden biomass include waste or by-products from forestry, such as thinning or residual slash from harvested operations, bark, wood chips, saw dust or the like. Further example of lignocellulose containing material include solid or liquid effluents or by-products from pulp and paper industry, such as pulpwood, wood hydrolysates of different origin and in different states of processing; mill residue, paper waste, such as waste from the production or recycling of newspapers, magazines, photocopying and computer printer papers and paper based packaging. Preferred lignocellulose containing material include spent liquor or waste liquor from pulping, such as acidic waste liquor, acidic sulphite waste liquor, neutralized waste liquor etc, including combinations thereof, such as mixed waste streams. Other waste material includes urban wood waste, logging slash, unmerchantable trees.

Other relevant lignocellulose containing material include non-wood plant material including stem, stalk, shrub, hulls, foliage, fibre, shell, root, straw, hay, grass, reed or the like. Sources of straw include in particular cereals and cereal grasses, such as oat, wheat, barley, rye, oat, maize, rice, sorghum, Indian millet, or particulates of grain as straw, hulls, husks, fiber, stems, shells, corn cobs, corn straw, com fiber, nutshells, almond shells, coconut shells, bagasse, cotton seed bran, and cotton seed skins.

Additional sources of lignocellulose containing material can be root-crops, such as beets and tubers. The above examples are intended for illustrative purposes only, and are not limiting the scope of the invention. Further example of lignocellulose containing material include solid or liquid effluents or by-products from food and feed industry, for example effluents or by-products containing cellulose, hemicellulose, sugar or starch; solid or liquid waste or by-products from agriculture; by-products from gardening such as garden refuse or other waste or by-product streams or their components comprising compounds that can be fermented. Further example of the lignocellulose containing material may be selected from sugar cane, bagasse and molasses.

Additional sources of lignocellulose containing material can be marine biomass including algae, seaweeds, water hyacinth, giant Kelps, micro algae such as Chlorella, Dunaliella, and Euglena.

The lignocellulose containing material may be any of the above-mentioned materials in treated or untreated from. A skilled person can implement possibly necessary pretreatment steps without inventive effort and without undue experimentation. It is further contemplated that other means of facilitating the degradation of substrates can be used in the process, examples including, but not limited to mechanical disruption, ultrasonication, or steam and high-pressure pre-treatments.

Fermentation medium, in addition to carbon source may comprise suitable nitrogen source. Example of suitable nitrogen sources include, but are not limited to, amino acids in either crude form or pure, proteins like yeast extract, dried yeast, ethanol-yeast distillate, meat extract, peptone, soya powder, soybean hydrolysate, malt extract, casein hydrolysate, or urea, fish meal, fish hydrolysate, corn steep liquor, cotton seed meal, urea, other nitrogen containing material including synthetic or inorganic sources of nitrogen in solid, liquid or gaseous forms.

Fermentation medium may be supplemented with suitable inorganic salts including but not limited to, salts of potassium, calcium, sodium, magnesium, iron, cobalt, zinc, copper, molybdenum, tungsten, other trace elements and phosphoric acid.

The suitable examples of trace nutrients, growth factors, and the like that may be added in the medium include, but are not limited to, coenzyme A, pantothenic acid, pyridoxine-HCI, biotin, thiamine, riboflavin, flavine mononucleotide, flavine adenine dinucleotide, DL-6,8-thioctic acid, folic acid, Vitamin B12, other vitamins, amino acids such as cysteine and hydroxyproline, bases such as adenine, uracil, guanine, thymine and cytosine, sodium acetate, and the like, either as pure or partially purified chemical compounds or as present in natural materials.

In method of the present invention the pH of the starting materials is adjusted to the range of about pH 5-6.5, preferably 5.5-6. The pH may be adjusted by the addition of appropriate amounts of pH regulators, such as acids or alkali according to well-known procedures. The fermentation is performed in a temperature interval of about 25 to 3O.degree. C, preferably about 28 to about 3O.degree. C. Other fermentation conditions include carrying out the fermentation under agitation, aerobic condition. Fermentation may be carried out for the period of about at least 20 hours preferably 24-30 hours.

The process according to the invention can be performed as a batch fermentation, wherein the microorganisms are killed or otherwise discarded after the fermentation. In another embodiment of the invention the fermentation process is performed as a continuous or semi-continuous process, where starting materials and/or nutrients are added during fermentation.

The present invention strain Saccharomyces cerevisiae MTCC 5452, has capability to tolerate high concentrations of xylose of about 20% and glucose of about 18%. Such characteristic advantageously allows the Saccharomyces cerevisiae MTCC 5452 strain of the present invention to ferment xylose and glucose at higher concentrations. Such ability may allow the Saccharomyces cerevisiae MTCC 5452 strain to produce ethanol with higher productivity, particularly in continuous mode fermentation.

To obtain the ethanol after the fermentation, the biomass first needs to be separated from the fluids by means such as centrifugation or sedimentation. Subsequently, the ethanol can be separated from the biomass by any conventional method, such as distillation, membrane separation, enzyme process and gasification.

The fermentative method of the present invention by Saccharomyces cerevisiae MTCC 5452 strain allows the ethanol to be produced with the volumetric productivity of at least 3 g/l/h and in some of the embodiments as high as 4.2 g/l/h. The yield that can be obtained may be atleast 75%, in some of the embodiments, it may be 75-90%.

Advantages of the Invention: It is known to the person skilled in the ordinary art that naturally occurring Saccharomyces cerevisiae ferments only C-6 sugars and cannot and does not ferment C-

5 sugars. This necessitates the need of complex and lengthy procedures such as multiple stage culture, use of mix culture or modifying the organism by mutation or recombinant techniques to ferment C- 5 sugar and produce ethanol. It was surprisingly found by the inventors of the present invention that a novel strain Saccharomyces cerevisiae MTCC 5452 of the present invention unlike the conventionally available wild type strains of Saccharomyces cerevisiae has the capability of fermenting C-5 sugar either alone or with C-6 sugars. The present invention method produces ethanol with higher productivity without having a need to carry out the fermentation in multiple steps and/or mixed culture of microorganisms and/or genetic modification or mutation of the conventionally available strain. The additional advantage is the possibility of utilizing of C-5 sugar in particular xylose comprised in low cost lignocellulose containing material including waste materials of different kinds which were difficult to be used with available conventional or genetically modified Saccharomyces cerevisiae strains. Since it has high tolerance capacity towards C-5 sugars, C-6 sugars, as well as ethanol, the strain can utilize starting materials that is hemicellulose and/or cellulose substrates with higher concentration of C-5 and/or C-6 sugars, as well as provide higher ethanol productivity. The Saccharomyces cerevisiae MTCC 5452 capable of utilizing C-5 as well C-

6 sugar make the ethanol producing process efficient with productivity as high as 5 g/l/h.

Further aspects of the invention, and the advantages associated therewith, will be evident to a skilled person upon study of the description, examples and claims. The present invention will now be described in the following non-limiting examples. While the present invention has been described herein with respect to the various exemplary embodiments, it will be apparent to the person ordinary skilled in the art that various modifications, variations and sub combinations of the embodiments can be made to the within the scope of the invention.

EXAMPLES

EXAMPLE 1

Inoculum preparation:

The Saccharomyces cerevisiae MTCC 5452 strain was isolated from soil and first grown onto agar slants. The microorganism was then inoculated into broth each containing 10 -15ml of Sabouraud's media. The tubes were incubated at a temperature ranging from 25-3O⁰C for 24-36 hours in a shaker at 150 rpm and the pH is kept in the range of 5.5 - 6.5 The inoculum thus obtained was used for tolerance tests as well as for fermentation for producing ethanol.

EXAMPLE 2

Xylose Tolerance Test of Saccharomyces cerevisiae MTCC 5452:

Tolerance test of Saccharomyces cerevisiae MTCC 5452 towards xylose was carried out as per the known method. For the tolerance test 20% Xylose in Sabouraud broth was used as stock solution and Sabouraud broth without xylose as a diluent. Each of the test tube received 0.1 ml of inoculum Saccharomyces cerevisiae MTCC 5452 and total volume was adjusted to 10 ml using stock solution and diluent as given in Table 2 to get the desired serial dilution. The negative control tube did not receive any inoculum. The test tubes were incubated at room temperature for period of 48 hours, observations were noted in Table 2. Table 2:

Legend:

+ ve: Growth observed - ve: No growth observed. Observation and results: The above observations indicate that the Saccharomyces cerevisiae MTCC 5452 not only has capability of fermenting xylose but also tolerates xylose upto 20% concentration.

EXAMPLE 3

Sugar Tolerance Test for Glucose:

Tolerance test of Saccharomyces cerevisiae MTCC 5452 towards glucose was carried out as per the known method. For the tolerance test 20% glucose in Sabouraud broth was used as stock solution and Sabouraud broth without glucose as a diluent. Each of the test tube received 0.1 ml of inoculum Saccharomyces cerevisiae MTCC 5452 and total volume was adjusted to 10 ml using stock solution and diluent as given in Table 3 to get the desired serial dilution. The positive control did not receive any diluent and negative control tube did not receive any inoculum. The test tubes were incubated at room temperature for period of 24 hours; observations were noted in Table 3.

Table 3:

Legend: + : Growth -: No growth.

The above observations indicate that the Saccharomyces cerevisiae MTCC 5452 tolerates glucose upto 18 % concentration.

EXAMPLE 4

Production of ethanol from sugarcane bagasse hydrolyzed by hydrothermal treatment:

The Saccharomvces cerevisiae MTCC 5452 10% inoculum as obtained in Example 1 was used for fermentation of hemicellulose hydrolysate rich in xylose and obtained by sugarcane bagasse hydrolyzed by hydrothermal treatment . 10 ml of inoculum was added to 250 ml flask containing 90 ml of fermentation broth containing hemicellulose hydrolysate as mentioned above and incubated at 24⁰C for 26 hrs. After fermentation, ethanol was recovered by distillation method and analyzed by GC (ethanol concentration - 56%), the productivity was 3.5 g/l/h and yield was 76%, as depicted in Fig. 1.

EXAMPLE 5

Production of ethanol from hemicellulose hydrolysate: The Saccharomvces cerevisiae MTCC 5452 10% inoculum as obtained in Example 1 was used for fermentation of hemicellulose hydrolysate rich in xylose, obtained by acid hydrolysis of hemicellulose fraction of sugarcane bagasse. 100ml Inoculum was added to 5000 ml flask containing 900 ml fermentation broth containing hemicellulose hydrolysate as mentioned above and incubated at 26⁰C for 28 hrs.

After fermentation, ethanol was recovered by distillation method and analyzed by GC (ethanol concentration 55%), the productivity was 3.8 g/l/h and the yield was 83%, as depicted in Fig. 2.

EXAMPLE 6

Production of ethanol from sugarcane bagasse hydrolyzed by steam explosion:

The Saccharomvces cerevisiae MTCC 5452 10% inoculum as obtained in Example 1 was used for fermentation of hydrolysate containing C-5 and C-6 sugars obtained by sugarcane bagasse hydrolyzed by steam explosion. 150ml Inoculum was added to 5000 ml flask containing 1350 ml fermentation broth containing hydrolysate as mentioned above and incubated at 27⁰C for 30 hrs. After fermentation, ethanol produced was obtained by distillation and analyzed by GC (ethanol concentration 56%). The productivity was 4.2 g/l/h and the yield was 90%, as depicted in Fig. 3.

Although the invention has been described with regard to its preferred embodiments, which constitute the best mode presently known to the inventors, it should be understood that various changes and modifications as would be obvious to one having the ordinary skill in this art may be made without departing from the scope of the invention which is set forth in the claims appended hereto.

Claims

1. A microorganism Saccharomyces cerevisiae strain MTCC 5452, being capable of fermenting C-5 sugar and producing ethanol.

2. The microorganism strain as claimed in claim 1, wherein the Saccharomyces cerevisiae strain MTCC 5452 is further capable of fermenting C-6 sugar.

3. The microorganism strain as claimed in claim 1, wherein the Saccharomyces cerevisiae strain MTCC 5452 is capable of tolerating about 20 % of the xylose concentration in the fermentation medium.

4. The microorganism strain as claimed in claim 1, wherein the Saccharomyces cerevisiae strain MTCC 5452 is capable of tolerating about 18 % of the glucose concentration in the fermentation medium.

5. A culture consisting essentially of a Saccharomyces cerevisiae strain MTCC 5452 of claim 1.

6. A method for producing ethanol comprising; fermenting a medium comprising atleast a carbon source with a Saccharomyces cerevisiae strain MTCC 5452.

7. The method as claimed in claim 6, wherein the carbon source is a C- 5 sugar and/or C-6 sugar.

8. The method as claimed in claim 7, wherein the C-5 sugars are comprised in hemicellulose hydrolysate and/or cellulose hydrolysate.

9. The method as claimed in claim 8, wherein the C-6 sugars are comprised in hemicellulose hydrolysate and/or cellulose hydrolysate.

10. The method as claimed in claim 8 or 9, wherein the hemicellulose hydrolysate and cellulose hydrolysate are derived from lignocellulose containing material.

11. The method as claimed in claim 10, wherein the lignocellulose containing material is hydrolyzed before and/or during fermentation.

12. The method as claimed in claim 11, wherein the lignocellulose containing material is hydrolyzed by at least one process selected from the group consisting of autohydrolysis, enzymatic hydrolysis with enzymes having a hemicellulolytic or cellulolytic activity, or acid hydrolysis.

13. The method as claimed in claim 12, wherein the autohydrolysis treatment is selected from atleast one process selected from hydrothermal treatment or steam explosion.

14. The method as claimed in claim 11, wherein the lignocellulose containing material is pre-treated before and/or during hydrolysis.

15. The method as claimed in claim 7, wherein the C-5 sugar is selected from groups consisting of xylose, arabinose, ribose and other monosaccharides having C-5 carbon that can be fermented by Saccharomyces cerevisiae strain MTCC 5452, preferably it is xylose.

16. The method as claimed in claim 7, wherein the C-6 sugars are selected from the group consisting of glucose, fructose, galactose, rhamnose, mannose, and other monosaccharides.

17. The method as claimed in claim 6, wherein the fermentation is aerobic fermentation.

18. The method as claimed in claim 6, wherein the fermentation is carried out as batch fermentation.

19. The method as claimed in claim 6, wherein the fermentation is carried out as a continuous or semi-continuous process.

20. The method as claimed in claim 6, wherein the fermentation is carried out at pH 5-6.5.

21. The method as claimed in claim 6, wherein the fermentation is carried out at a temperature of about 24 to about 30.degree. C.

22. A method for producing ethanol as substantially described herein above and illustrated by drawings and example.