FIELD OF THE INVENTION

The invention relates to a modified strain of yeast useful for the production of alcohol and a process for the preparation of the modified strain of yeast.

BACKGROUND OF THE INVENTION

It is well known that alcohol can be obtained by fermentation of molasses by employing certain microbes like yeast capable of carrying out the fermentation process.

Yeasts are eukaryotic micro-organisms classified in the kingdom Fungi. Most of the yeasts reproduce asexually by budding and few of them reproduce by binary fission. The useful physiological properties of yeast have led to their use in the field of biotechnology. Fermentation of sugars by yeast is the oldest and largest application of this technology. The most common species used for preparation of alcohols are Saccharomyces cerevisiae and S. carlsburgiensis, Kluyveromyces marxianus and Saccharomyces uvarum

Schizosaccharomyces pombe:

- Kingdom: Fungi

- Phylum: Ascomycota

- Subphylum: Taphrinomycotina

- Class: Schizosaccharomycetes

- Order: Schizosaccharomycetales

- Family: Schizosaccharomycetaceae

- Genus: Schizosaccharomyces

It is also called as "fission yeast". It is used as a model organism in molecular and cell biology. It is a unicellular eukaryote, whose cells are rod-shaped. Typically, cells measure 3 to 4 micrometers in diameter and 7 to 14 micrometers in length. Its genome, which is approximately 14.1 million base pairs, is estimated to contain 4,970 protein-coding genes and at least 450 non-coding RNAs.

These cells maintain their shape by growing exclusively through the cell tips and divide by medial fission to produce two daughter cells of equal sizes, which makes them a powerful tool in cell cycle research. The sequence of the S. pombe genome was published in 2002, by a consortium led by the Sanger Institute, becoming the sixth model eukaryotic organism whose genome has been fully sequenced. This has fully unlocked the power of this organism, with many genes homologous to human disease genes being identified. In 2006, sub-cellular localization of all the proteins in S. pombe was published using green fluorescent protein as a molecular tag. S. pombe has also become an important organism in studying the cellular responses to DNA damage and the process of DNA replication.

EXISTING KNOWLEDGE

Following patents/ patent applications discloses various yeast strains useful in the preparation of alcohols.

US Patent No. 5693526 discloses a strain of the yeast Saccharomyces cerevisiae having accession number MTCC Y0022B211 (=NCYC 2647) which is useful for the preparation of ethanol by fermentation of sugars.

US Patent No. 4910144 discloses a yeast strain (M-9 (FERM BP 1481 of the genus Saccharomyces cerevisiae) useful in producing alcohol from beet molasses by fermentation.

US Patent No. 5858764 discloses Saccharomyces cerevisiae yeast strain useful for fermentation of sugars and a process to obtain such yeast.

US20090226993 discloses ethanol producing thermophilic yeast strain (Kluyveromyces IIPE453 MTCC 5314) which is ascomycetous yeast of the fungal family saccharomycetaceae.

WO/2008/062558 discloses yeast strain (Kluyveromyces marxianus NITE BP-283) capable of producing ethanol through fermentation.

The main drawback of conventional yeast strains is their failure to grow and remain active at high sugar concentrations and at temperature up to 34 C during the fermentation process that produces ethanol. At the same time, unless a high sugar concentration is used in the fermentation process, it is not possible to obtain an increased level of ethanol. The cumulative effect of these problems in the conventional process of production of ethanol is a low level of ethanol produced in the wash. Therefore, the processes are not efficient and also not economical. Accordingly it is desirable to develop a new yeast strain which withstand high dissolved solids and remain active at high sugar concentration and at temperature up to 34° C during the fermentation process.

OBJECTS OF THE INVENTION

It is an object of the present invention to provide a yeast strain for continuous fermentation process.

It is another object of the present invention to provide a yeast strain which withstands high dissolved solids.

It is still another object of the present invention to provide a yeast strain which grow and remain active at high sugar concentrations and at temperature up to 34° C during the fermentation process.

It is yet another object of the present invention to provide a yeast strain which survive at a temperature of about 32 to about 34°C.

It is a further object of the present invention to provide a yeast strain which effects the fermentation with high efficiency and produces alcohol in high amount.

SUMMARY OF THE INVENTION

In accordance with the present invention there is provided a process for the preparation of a modified strain of the yeast Schizosaccharomyces pombe; said process comprising the following steps:

a) inoculating yeast culture of Schizosaccharomyces pombe (Accession No. 3360) in a solution selected from the group consisting of saline solution and ringer salt solution to obtain a primary culture and subjecting the same to serial dilutions to obtain serially diluted cultures comprising yeast strain;

b) preparing Sabouraud agar medium and aseptically transferring the medium to sterilized Petri plates followed by sterilizing the Petri plates with the medium;

c) inoculating the serially diluted yeast strain on Sabouraud agar medium present in the Petri plates under aseptic condition using streak plate method;

d) incubating the Petri plates at 32°C for 35 to 48 hours;

e) sub-culturing the incubated yeast;

f) inoculating the sub-cultured yeast on sterilized molasses medium in a first propagation vessel wherein the concentration of sugar is maintained at 5% w/v to obtain a first biomass;

g) transferring the first biomass upon reduction of sugar concentration to about 1.5 to about 1.8 % to a second propagation vessel which contains a molasses medium with a sugar concentration of about 5% w/v followed by introduction of nutrients to obtain a second biomass;

h) transferring the second biomass upon reduction of sugar concentration to about 1.5 to about 1.8 % to a third propagation vessel which contains a molasses medium with a sugar concentration of about 5% w/v followed by introduction of nutrients to obtain a third biomass;

i) transferring the third biomass upon reduction of sugar concentration to about 1.5 to about 1.8 % to a fourth propagation vessel which contains molasses medium with a sugar concentration of about 5% w/v w/v followed by introduction of nutrients to obtain a fourth biomass;

j) transferring the fourth biomass to yeast activation tank containing molasses medium followed by introduction of air through the medium and maintaining the sugar concentration within the range of about 7% to about 10 % w/v followed by introduction of nutrients to obtain a biomass enriched with yeast cells;

k) inoculating the biomass enriched with yeast cells on molasses medium with a sugar concentration of 10 to 12 % w/v followed by introduction of air and nutrients;

I) isolating the yeast cells and inoculating the same on Sabouraud agar medium by using streak plate method to obtain a modified yeast strain of Schizosaccharomyces pombe.

Typically, the solution is serially diluted in a range between 10"1 to 10" .

Typically, the ringer salt solution is prepared by dissolving sodium chloride (8.5 gm), potassium chloride (0.2 gm), calcium chloride (0.2 gm) and sodium bicarbonate( 0.01 gm ) in water.

Typically, the Sabouraud agar medium comprises 40gm/l glucose, 10gm/l mycological peptone, 15gm/l agar and distilled water.

Typically, the pH of the Sabouraud agar medium is about 5.6 ±0.2. Typically, the pH of the molasses medium is about 4.3 to about 4.5.

Typically, the molasses medium further comprises 0.30 % of urea, 0.15 % of diammonium phosphate, 0.15 % of magnesium sulphate and 0.0004 % of sodium metabisulphite.

Typically, the volume of molasses medium in the second, third and fourth propagation vessel is about 70 %.

In accordance with another aspect of the present invention there is provided a modified strain of Schizosaccharomyces pombe yeast that remains stable at 12 to 14 % dissolved solid and which withstands the temperature of about 32 to about 34 °C.

Typically, the strain is rod shaped with an average size of about 5u to 9u.
Typically, the strain is capable of growing in high sugar concentration (12.5 to 13.5 %) without any flocculation.

Typically, the strain is capable of withstanding high osmotic pressure without changing the morphology.

In accordance with another aspect of the present invention there is provided a method for producing alcohol; said method comprising fermenting at least one substrate selected from the group consisting of beet molasses, cane molasses, sweet potato, potato, casaba, rice, agricultural waste and hydrolyzates thereof using a modified Schizosaccharomyces pombe yeast stain of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the present invention there is provided a process for the preparation of a modified strain of the yeast Schizosaccharomyces pombe. The process comprising the following steps:

In the first step, yeast culture of Schizosaccharomyces pombe (Acession No. 3360) is inoculated in a saline solution or ringer salt solution to obtain a primary culture which is then subjected to serial dilutions to obtain serially diluted cultures comprising yeast strain.

Typically, the ringer salt solution is prepared by dissolving sodium chloride ( 8.5 gm), potassium chloride (0.2 gm), calcium chloride (0.2 gm) and sodium bicarbonate( 0.01 gm ) in water. Typically, the solution is serially diluted in a range between 10"' to 10'9.

In the next step, Sabouraud agar medium is prepared by dissolving 40gm of glucose, lOgm of mycological peptone and 15gm of agar in 1000 ml of distilled water. The pH of the Sabouraud agar medium is maintained to 5.6 ±0.2. The medium is then aseptically transferred into sterilized Petri plates. The Petri plates containing medium are sterilized further.

The serially diluted yeast strain is then inoculated on sterilized Sabouraud agar medium present in the Petri plates under aseptic condition using streak plate method. The plates are then kept in an incubator for incubation at 32°C for 35 to 48 hours. The obtained yeast is then subjected to sub-culturing.

The sub-cultured yeast is inoculated on sterilized molasses medium in a first propagation vessel wherein the concentration of sugar is maintained at 5% w/v to obtain a first biomass. The first biomass upon reduction of sugar concentration to about 1.5 to about 1.8 % is transferred to a second propagation vessel which contains a molasses medium with a sugar concentration of about 5% w/v and nutrients are introduced to obtain a second biomass. The second biomass upon reduction of sugar concentration to about 1.5 to about 1.8 % is transferred to a third propagation vessel which contains a molasses medium with a sugar concentration of about 5% w/v and nutrients are introduced to obtain a third biomass. The third biomass upon reduction of sugar concentration to about 1.5 to about 1.8 % is transferred to a fourth propagation vessel which contains molasses medium with a sugar concentration of about 5% w/v and nutrients are introduced to obtain a fourth biomass.

In the next step, the fourth biomass is transferred to yeast activation tank containing molasses medium followed by introduction of air through the medium and maintaining the sugar concentration within the range of about 7% to 10 % w/v and nutrients are introduced to obtain a biomass enriched with yeast cells. The obtained biomass enriched with yeast cells is inoculated on molasses medium with a sugar concentration of 10 to 12 % w/v followed by introduction of air and nutrients. Finally, the yeast cells are isolated and inoculated on Sabouraud agar medium by using streak plate method to obtain a modified yeast strain of Schizosaccharomyces pombe.

Typically, the pH of the molasses medium is about 4.3 to about 4.5.

In accordance with another embodiment of the present invention the molasses medium further comprises 0.30 % of urea, 0.15 % of diammonium phosphate, 0.15 % of magnesium sulphate and 0.0004 % of sodium metabisulphite.

Typically, the volume of molasses medium in the second, third and fourth propagation vessel is about 70 %.

In accordance with another aspect of the present invention there is provided a modified strain of Schizosaccharomyces pombe yeast.

In accordance with the present invention the Schizosaccharomyces pombe yeast used for the preparation of modified strain has following characteristics:

• Growth temperature: 28-29°C

• Withstands only 8% dissolved solids

The modified strain of Schizosaccharomyces pombe yeast in accordance with the present invention has following characteristics:

• The strain is rod shaped with an average size of about 5u to 9u.

• The strain grows in a normal fashion in high sugar concentration (12.5 to 13.5 %) without any flocculation.

• The strain grows in high dissolved solids for longer period of time without changing the morphology i.e. it is stable at 12 to 14 % dissolved solid.

• The strain bears high osmotic pressure without changing the morphology

• It withstands the temperature of about 32 to about 34 °C.

In accordance with another aspect of the present invention there is provided a method for producing alcohol; said method comprising fermenting at least one substrate selected from the group consisting of beet molasses, cane molasses, sweet potato, potato, casaba, rice, agricultural waste and hydrolyzates thereof using a modified Schizosaccharomyces pombe yeast stain of the present invention.

While considerable emphasis has been placed herein on the specific features of the preferred embodiment, it will be appreciated that many additional features can be added and that many changes can be made in the preferred embodiment without departing from the principles of the invention. These and other changes in the preferred embodiment of the invention will be apparent to those skilled in the art from the disclosure herein, whereby it is to be distinctly understood that the foregoing descriptive matter is to be interpreted merely as illustrative of the invention and not as a limitation.

We Claim:

1. A process for the preparation of a modified strain of the yeast Schizosaccharomycespombe; said process comprising the following steps:

a. inoculating yeast culture of Schizosaccharomyces pombe (Accession No. 3360) in a solution selected from the group consisting of saline solution and ringer salt solution to obtain a primary culture and subjecting the same to serial dilutions to obtain serially diluted cultures comprising yeast strain;

b. preparing Sabouraud agar medium and aseptically transferring the medium to sterilized Petri plates followed by sterilizing the Petri plates with the medium;

c. inoculating the serially diluted yeast strain on Sabouraud agar medium present in the Petri plates under aseptic condition using streak plate method;

d. incubating the Petri plates at 32°C for 35 to 48 hours;

e. sub-culturing the incubated yeast;

f. inoculating the sub-cultured yeast on sterilized molasses medium in a first propagation vessel wherein the concentration of sugar is maintained at 5% w/v followed by introduction of nutrients to obtain a first biomass;

g. transferring the first biomass upon reduction of sugar concentration to about 1.5 to about 1.8 % to a second propagation vessel which contains a molasses medium with a sugar concentration of about 5% w/v followed by introduction of nutrients to obtain a second biomass;

h. transferring the second biomass upon reduction of sugar concentration to about 1.5 to about 1.8 % to a third propagation vessel which contains a molasses medium with a sugar concentration of about 5% w/v followed by introduction of nutrients to obtain a third biomass.

i. transferring the third biomass upon reduction of sugar concentration to about 1.5 to about 1.8 % to a fourth propagation vessel which contains molasses medium with a sugar concentration of about 5% w/v followed by introduction of nutrients to obtain a fourth biomass.

j. transferring the fourth biomass to yeast activation tank containing molasses medium followed by introduction of air through the medium and maintaining the sugar concentration within the range of about 7% to about 10 % w/v followed by introduction of nutrients to obtain a biomass enriched with yeast cells;

k. inoculating the biomass enriched with yeast cells on molasses medium with a sugar concentration of 10 to 12 % w/v followed by introduction of air and nutrients;

l. isolating the yeast cells and inoculating the same on Sabouraud agar medium by using streak plate method to obtain a modified yeast strain of Schizosaccharomyces pombe.

2. The process as claimed in claim 1, wherein the solution is serially diluted in a range between 10"1 to 10"9.

3. The process as claimed in claim 1, wherein the Sabouraud agar medium comprises 40gm/l glucose, l0gm/1 mycological peptone, 15gm/l agar and distilled water.

4. The process as claimed in claim 1, wherein the pH of the Sabouraud agar medium is about 5.6 ± 0.2.

5. The process as claimed in claim 1, wherein the pH of the molasses medium is about 4.3 to about 4.5.

6. The process as claimed in claim 1, wherein the molasses medium further comprises 0.30% of urea, 0.15% of diammonium phosphate, 0.15% of magnesium sulphate and 0.0004% of sodium metabisulphite.

7. The process as claimed in claim 1, wherein the volume of molasses medium in the second, third and fourth propagation vessels is about 70 %.

8. A modified strain of Schizosaccharomyces pombe yeast; that remains stable at 12 to 14 % dissolved solid and which withstands the temperature of about 32 to about 34 °C.

9. The strain as claimed in claim 1, wherein the strain is rod shaped with an average size of about 5u to 9u.

10. The strain as claimed in claim 1, wherein the strain is capable of growing in high sugar concentration (12.5 to 13.5 %) without any flocculation.

11. The strain as claimed in claim 1, wherein the strain is capable of withstanding high osmotic pressure without changing the morphology.

12. A method for producing alcohol; said method comprising fermenting at least one substrate selected from the group consisting of beet molasses, cane molasses, sweet potato, potato, casaba, rice, agricultural waste and hydrolyzates thereof using Schizosaccharomyces pombe yeast stain as claimed in claim 8.