Title of Invention	A PROCESS FOR THE PREPARATION OF IMPROVED THEREMOTOLERANT FLOCCULENT SACCHAROMYCES
Abstract	The present invention relates to process of preparation of thermotolerant satrains of the genus Saccharomyces species - cerevisiae by using the genetic engineering, mutation, enrichment and repeated selection processes from existing diploid homothallic strains. The invention also relates to a process of high temperature fermentation using these genetically modified strains wherein the ethanol so prepared is useful both for potable and industrial purposes,

Title of Invention

A PROCESS FOR THE PREPARATION OF IMPROVED THEREMOTOLERANT FLOCCULENT SACCHAROMYCES

Abstract

The present invention relates to process of preparation of thermotolerant satrains of the genus Saccharomyces species - cerevisiae by using the genetic engineering, mutation, enrichment and repeated selection processes from existing diploid homothallic strains. The invention also relates to a process of high temperature fermentation using these genetically modified strains wherein the ethanol so prepared is useful both for potable and industrial purposes,

Full Text	1 The invention relates to a process for the preparation of improved thermotolerant focculent Saccharomyces. This invention particularly relates to an improved yeast strain of Saccharomyces cerevisiae useful for the preparation of ethanol by the fermentation of sugars. The ethanol so prepared is useful both for potable and industrial purposes. / It is well known that ethanol or ethyl alcohol (C2H5OH) is produced by both fermentation and synthetic methods. Fermentation techniques for ethanol production developed during the early part of this century were supplemented by synthetic processes based on crude petroleum, as oil was much cheaper and abundantly available. However, of late, it is realised that petroleum oil reserve is not going to last long and fermentative production of ethanol has again picked up, using various kinds of renewable ferrnemabie subsiraics be il (i) sugar (from sugar-cane, sugar beet, fruit) which may be converted to ethanol directly; (ii) starch (from grain, root crops) which are first hydrolysed to fermentable sugars by enzymes; and (iii) cellulose (from wood, agricultural wastes, etc.) which are converted to sugars. (Biotechnology: Economic and Social Aspects :- Issues for Developing Countries. Eds. : E.J. Da Silva, C.Ratledge and A. Sesson; Cambridge University Press, p24 1992). Ethanol production by fermentation is based mainly on yeasts, and for large scale fuel production these are generally of the genus Saccharomyces. The distillers all over the world would like to have a fermentation process which can run at a temperature range between 28°C and 38°C and gives high percentage of alcohol in the broth without sacrificing fermentation efficiency. The primary benefits of such an operation are : a) potential to increase the productivity (wherein productivity is defined as amount of alcohol produced per unit fermcnter vol./hr). b) fermenters may be operated at normal as well as higher temperature (28°C to 38°C) by design or caused by accidental failure of cooling device or electrical break down in the plant. c) saving in energy/steam requirement during distillation; energy being the major variable cost in distillery operation. d) reduction in the volume of effluent. e) in case of flocculent yeast strains, fermentation may be done by recycling of cells, in a continuous mode or by conventional batch rnnrji? Rer.ause of their flocculent nature, cells are easily separated from the fermented broth and in a recycling mode or in a continuous mode less sugar is expected to be required for building up of biomass. In conventional method of ethanol production, initial concentration of sugar in the broth is kept between 14 to 16 percent. Sugar concentrations higher than this is detrimental to growth of yeast strains used in conventional process and fermentation is thus affected. After completion of fermentation, 6.5 to 8 percent alcohol is obtained in the wash. Alcohol is then recovered by distillation using steam. In order to achieve higher ethanol level in the wash, initial sugar concentration in the broth should also be higher, thereby increasing the osmolarity of the medium which is detrimental to the growth and fermentation of the conventional yeast strains. Conventional strains are also sensitive to high level of ethanol in the broth and they do not have flocculent characteristic. One of the drawbacks in conventional yeast strains is their sensitivity to high temperature. High temperature in the fermenter is generated by ambient heat as well as metabolic activities of yeast strains. This necessitates efficient cooling device which may be expensive. In case of power failure or accidental break down of cooling device, fermentation does not get completed resulting in wastage of material and other resources. Moreover, conventional yeast strains fail to grow and become inactive at high sugar concentration during fermentation process. At the same time unless high sugar concentration is used in the fermentation process, it is not possible to obtain increased level of ethanol. Concurrent with this problem conventional yeasts will not be effective in the fermentation, if ethanol concentration is increased in the broth. The cumulative effects of these problems in the conventional process of production of ethanol leads to low level of ethanol in the wash and consequently, consumption of steam per litre of ethanol distilled is high. Therefore, the processes are not efficient and also not economical. Moreover, separation of conventional yeast strains from fermented broth is not easy and may not be very efficient in a cell-recycling or continuous mode of operation. Realising the need to have strains possessing various desirable properties and useful for high density fermentation and in a wide range of operating temperatures we continued our research with the objective of developing thermotolerant strains of yeast which, in addition to tolerating high initial sugar concentration, i.e. having osmotolerant characteristic, could Saccharomyces particularly species cerevisiae which are useful for fermentation pr^s^s for the preparation of ethanol. Another objective of the present invention is to introduce the flocculation characteristics in some of the said thermotolerant strains which are useful for the preparation of ethanol. Another objective of the present invention is to produce thermotolerant strains of the genus Saccharomyces species - cerevisiae by using the genetic engineering, mutation, enrichment and repeated selection processes. Another objective of the present invention is to introduce the flocculent characteristics in the thermotolerant strains of the genus- Saccharomyces species - cerevisiae by genetic hybridisation or cytoduction followed by selection. By thermotolerant it is meant that the strain should be able to efficiently ferment sugars to ethanol at a higher temperature (>35°C) than that is optimal for normal strains (28°C to 32°C), it should also retain good viability at the end of fermentation, so the cells can be reused in subsequent rounds of fermentation. One common process employed so far to obtain thermotolerant strains of yeast is to screen for such strains in the natural environment (D'Amore T et al., 1989, Enzyme Microb Technol 11: 411-416; Banat IM & Marchant R, 1995, World J. Microbiol Biotechnol 11: 304-306; Banat IM et al, 1992, World J. Microbiol Biotechnol 8: 259-263; Hacking AJ et al., 1984, Appl Microbiol. Biotech. 19: 361-363; Kida et al, 1992, J. Ferment & Bioeng. 74: 169-173; Laluce C et al, 1991, Biotechnol Bioeng 37: 528-536; Rosa MF et al, 1987, Biotech Lett. 9: 441-444). Though this process is simple, it can not obviously be used to improve pre-existing strains that may have many other desired features such as high osmotolerance, high ethanol tolerance and faster fermentation rate. One r^tbod often used to combine thermotolerance with other good properties is by protoplast fusion of strains having such properties (Kida et al. ,1992, J. Ferment & Bioeng. 74: 169-173; Seki T et al., 1983, Biotechnol Lett. 5: 351-356; Stewart GG et al., 1988, US patent No. 4,772,556). However, the disadvantage here is that unwanted features from the parent strains will also come into the hybrid strain and there is no method by which those can be readily removed from the hybrid. Thus the present invention relates to a process for the preparation of thermotolerant strains of yeast which obviates the drawbacks detailed above and thermotolerant mutants can be obtained starting from existing diploid homotnailic strains, and the mermotoierance property so obtained can be combined with good properties from other strains. The strains are prepared such that any undesirable features introduced inadvertently can also be readily removed. Distillery strains are usually diploid (having two sets of genes) or polyploid (having more than two sets of genes) and may be homothallic, i.e. haploid cells derived from spores can become diploid by mating with opposite mating-type (which originate in the population by switching over of mating-type). It is difficult to isolate mutants from diploid or polyploid strains since most of the mutations are recessive. That is, in a cell where normal gene and recessive mutant gene are present together, the former will mask the expression of the latter. It is, therefore, essential to generate stable haploid cells for generating mutant strains. Once desired mutants are obtained they can be further manipulated for construction of other strains with novel characteristics. In general, desirable genetic properties of two strains or more may be brought together, for creating novel straias by many ways such as, mating, (also known as crossing, genetic hybridisation), cytoduction, protoplast fusion as well as by genetic engineering and expression of the cloned gene(s). In doing this manipulations in the laboratory, the strains need to be properly marked and an innovative method has to be developed for the production of improved and novel strains. Accordingly, the present invention provides a process for the preparation of improved thermotolerant flocculent strains of Saccharomyces, which comprises a) growing a diploid homothallic strain of Saccharomyces, having features such as osmotolerance and ethanol tolerance, in a conventional medium, mutating the homothallic gene of the said strain by known methods, sporulating the resulting diploid and separating the spores to get stable haploids, b) treating the said haploids with conventional mutagen by known methods, and subjecting them to high temperature fermentation repeatedly, to get stable thermotolerant strains retaining all other properties of the parent strain such as osmotolerance and ethanol tolerance, c) growing a haploid strain having flocculation property using conventional medium, d) mating cells obtained in step (b) and (c) by known methods, e) isolating the resultant diploid cells by known methods, and checking for ploidy, prototrophy, antibiotic resistance and flocculation, f) sporulating the hybrid cells obtained in step (e) by known methods and separating individual spores from ascus using conventional methods, g) mating haploid cells obtained in step (f) with haploid cells of opposite mating type obtained in step (b) to get diploid cells having thermotolerant and flocculent characters, h) sporulating the cells obtained in step (g) and isolating the spores by known methods, and testing the resultant haploid cells for thermotnlerance; flocculation and other desirable properties. i) mating haploid cells obtained in step (h) with cells obtained in step (b), sporulating the resultant diploid and checking the resulting haploid cells for thermotolerance, flocculation and other desirable properties to get improved stable strains of thermotolerant and flocculent Saccharomyces useful for improved fermentation of fermentable sugars at high temperature. The homothallic strain of Saccharomyces cerevisiae used in step (a), deposited at Microbial Type Culture Collection and designated as MTCC Y0001, has the following properties: It is homothallic, diploid, prototrophic, osmotolerant, non-flocculent, and makes 10 to 12% (v/v) alcohol at 30°C, which is a subject matter of a copending patent application no. 748/DEL/93. This has been deposited at NCYC and has the accession no. NCYC 2646. The flocculent strain used in step (c), deposited at Microbial Type Culture Collection and designated as MTCC Y0002, and also having ATCC accession no. ATTC 90506, has the following properties: It is heterothallic, haploid, auxotrophic, flocculent, and produces 2 to 3 % (v/v) alcohol at 30°C. In a preferred embodiment the growth of MTCC Y0001 may be effected in a known medium such as YEPD (Yeast extract, Peptone, Dextrose) at a temperature in the range of 15°C to 35°C for a period of 1 to 10 days. In another preferred embodiment the HO gene in MTCC Y0001 may be mutated by conventional mutagenesis or by targeted gene disruption or by genetic hybridisation method. In yet another preferred embodiment the medium used for sporulating the diplcid strains such as Y0001 may consist of Agar, Yeast extract, Dextrose, Potassium acetate, and distilled water and the lytic enzyme used may be selected from lyticase, glusulase or zymolyase. In yet another preferred embodiment the mutagen used in step (b) may be selected from conventional chemical mutagens such as N-methyl-N'-nitro-N-nitrosoguanidine and ethyl methanesulfonate, or from ultraviolet light radiation, gamma radiation or similar agents. In yet another preferred embodiment fermentation may be carried out in a medium such as YEPD, YEPS (Yeast extract, Peptone, Sucrose), molasses or other media containing fermentable sugar at a temperature in the range of 15°Cto40°C. In yet another preferred embodiment the thermotolerant strains obtained in step (b) can be used as such for the fermentative production ^f ethanol. In yet another preferred embodiment the haploid strain MTCC Y0002 (ATCC 90506) used in step (c) may be selected from a haploid strain as such or haploid obtained from diploid strains. By way of example, other properly marked strains may be used. In yet another preferred embodiment the haploid strain in step (c) is grown in a known medium such as YEPD (Yeast extract, Peptone, Dextrose) at a temperature in the range of 15°C to 35°C for a period of 1 to 10 days. In yet another preferred embodiment the mating of cells obtained in steps (b) and (c) may be effected by cell-cell pairing or by mixing and plating on a suitable medium and incubating at a temperature in the range of 15°C to 37°C for a period in the range of 1 to 10 days. The medium used in step (e) may be selected from SD Medium (yeast nitrogen base, dextrose, agar and distilled water), YPD (Yeast extract, Peptone, Dextrose, Agar and distilled water) or YPG (Yeast extract, Peptone, Glycerol, Agar and distilled water). hi yet another preferred embodiment the medium used in step (e) for isolating diploids may be a conventional medium such as SDG (Yeast nitrogen base, Dextrose, Glycerol, Agar and distilled water) fortified with broad range antibiotic such as geniticin, oligomycin, chloramphenicol and the likes. In yet another preferred embodiment the different strains may separated by conver^cra! methods like streaking or dilution plating or by micromanipulation. In yet another preferred embodiment the improved thermotolerant flocculent strains may be either a haploid or diploid. The details of the steps of the process of the present invention are as follows: The parental strain designated as MTCC Y0001 is homothallic, diploid, prototrophic, osmotolerant, non-flocculent, and makes 10 to 12% v/v alcohol at 30°C. It can sporulate and produce haploid spores of a and alpha mating types. These spores spontaneously become diploids by switching mating types, and by subsequent hybridisation of cells of opposite mating types. To obtain stable haploid derivatives, the HO gene ( haploid gene ) of this strain, responsible for homothallism or persistent diploidy, was replaced by a mutated version (ho::neo, where it is disrupted by neo gene conferring resistance to antibiotic G418; van Zyl et al, 1993, Current Genetics 23: 290-294) by DNA mediated transformation. The transformants were selected on G418 containing medium. One of the resulting strains designated as T2 (Transformant no. 2) was sporulated on appropriate medium and spores (four per ascus) were separated with the help of a micromanipulator. Two of these four spores were sensitive to the antibiotic G418 and became diploid. The other two spores which were resistant to the said antibiotic and remained haploid were designated as T2-2A and T2-2C. While T2-2A is of a mating type, T2-2C is of alpha mating type. From these two strains respiratory deficient mutants which can not grow on glycerol were selected and designated as T2-2A-0 and T2-2C-0 respectively. One of the strains, T2-2C-0, was crossed with the heterothallic strain YNN217 (Ramer et al, 1992, Proc. Natl. Acad. Sci. 89: 11589-11593; obtained from Prof. Ronald Davis, Stanford University, USA) and diploids were selected. The selective medium was such that the original two strains (T2-2C-0 and YNN217) should not survive. Diploids only incorporating features of both the parents would be able to grow. A diploid strain was sporulated and the spores were separated as stated above. Two of the resulting four spores were resistant to G418 while the other two were sensitive and carried the ho gene. A G418 sensitive haploid strain was crossed with T2-2C-0 described above to produce a diploid strain. This diploid strain was sporulated, spores were separated and analysed for desired characteristics. The baplnid strain with desired characteristics was crossed again (back crossing) with T2-2C-0 and sporulated and spores analysed. This back crossing was done a total of six times to ensure retention of all the desired properties and finally two haploid strains 38-2A and 38-2C, having the ho gene ( haploid gene) from strain YNN217, and of mating types alpha and a, respectively, were selected. Two strains T2-2C and 38-2C described above were mutagenised with ethyl methane sulfonate and the mutagenised population was subjected to selection at high temperature fermentation in YEPS (Yeast extract, Peptone, Sucrose 18% w/v). After fermentation was complete, a portion of the cells were inoculated to fresh YEPS and fermentation was carried out as described above. This process was repeated six times and cells from last round of fermentation was plated out and individual colonies were tested for fermentation in YEPS at 38°C. Two strains designated as (MTCC Y0045) and (MTCC Y0046) derived from T2-2C and 38-2C, respectively, were finally selected. These two strains are stable haploid, thermotolerant, produce more ethanol at 38°C and retain higher viability at 38°C compared to the original parent strain MTCC Y0001. Genetic analysis showed that strain MTCC Y0045 is of alpha mating type and strain MTCC Y0046 belongs to a mating type. Mutations in these strains are in two different genes but both of them are recessive to wild type. Diploid versions of these haploid thermotolerant strains were also constructed by genetic crosses. Strain MTCC Y0046 (a mating type) was crossed with a strain 38-2A (alpha mating type and non thermotolerant). The resulting diploid strains were sporulated and spore clones were analysed as stated earlier. A thermotolerant clone of alpha mating type (106-2A) was selected and mated with MTCC Y0046. The resulting diploid strain designated as MTCC Y0047 was found to have high temperature fermentation ability comparable to haploid MTCC Y0046. In another strategy, spores of MTCC Y0001 were separated individually with the help of a micromanipulator and cells of MTCC Y0046 were placed next to these spores for mating, which was monitored under a microscope. The resulting diploids were sporulated and spores from individual ascus were allowed to form colonies on appropriate medium. Some of these haploid spores which received HO allele from MTCC Y0001 will switch mating type and thus generate diploid cells in the population. Few such diploids were analysed. One strain designated as MTCC Y0048 was found to have thermotolerance and high temperature fermentation efficiency comparable to original MTCC Y0046 haploid strain. In order to construct further improved strains having characteristics such as osmotolerance, ethanol tolerance, flocculation and capable of fermentation at high temperature, haploid strain MTCC Y0046 was crossed with another haploid strain designated as MTCC Y0002 as described earlier. Cells of these two haploid strains were placed next to each other in pairs, incubated till colonies appeared on plates and the colonies were then streaked on non-selective medium. Single colonies were picked and checked for desirable properties on appropriate selective medium. The selective medium was designed such that the original two strains (MTCC Y0046 and MTCC Y0002) would not survive. Only hybrids creaied by transfer of desirable genetic material from both MTCC Y0046 and MTCC Y0002 would be able to grow, and there were many such hybrids. The hybrids were sporulated and the spores were then initially checked for flocculation, nutritional requirements, thermotolerance and mating type. Some of them showed good flocculation, some showed mild flocculation while others did not flocculate. One of the spore clones designated as TF2-9C having flocculent, thermotolerant, high temperature fermentation properties and is of alpha mating type was mated with MTCC Y0046. A diploid strain designated as TF5 having desired properties was chosen. The strain TF5 was sporulated and the spores were analysed and one spore clone named TF5-5B having desired characteristics was crossed with MTCC Y0046 and a diploid strain designated as TF6 was obtained. TF6 was again sporulated and after separation of individual spores and their characterisation, a strain MTCC V0049 was selected. This strain is haploid, thermotolerant, flocculent and is efficient in fermentation at high temperature. The improved strains have been deposited in the National Facility on Microbial Type Culture Collection and Gene Bank (MTCC) located at the Institute of Microbial Technology, a constituent laboratory of Council of Scientific and Industrial Research. They have been assigned the following accession numbers: MTCC Y0045 haploid, thermotolerant, mutant derivative of T2-2C MTCC Y0046 haploid, thermotolerant, mutant derivative of 3 8-2C MTCC Y0047 diploid, therrrotolerant, derived from 106-2AxY0046 MTCC Y0048 diploid,, thermotolerant, derived from Y0046xYOOO1 MTCC Y0049 haploid, thermotolerant, flocculent derived from TF6 The improved strains listed above have the following characteristics: (i) they grow at a temperature ranging between 15°C and 38°C in Yeast Extract Peptone Dextrose (YEPD) medium with 2% glucose, (ii) they grow on agar plates containing molasses concentration of up to 50% and do not need any supplementation when grown in this medium, (iii) they produce 7% to 11.6% v/v ethanol at a temperature range between 28°C and 38°C with up to 90% efficiency, (iv) they also grow in Yeast Extract, Peptone, Dextrose (YEPD) medium in presence of up to 12% ethanol. The strains are, therefore, osmotolerant, ethanol tolerant, thermotolerant and produce high level of alcohol at temperature between 28°C and 38°C. v) in liquid medium, like Yeast Extract, Peptone, Dextrose or Yeast Extract Peptone Sucrose (YEPS), molasses or the like MTCC Y0049 shows flocculation. vi) MTCC Y0047 and MTCC Y0048 sporulate and produce ascospores. vii) they retain their viability and fermentation ability for use in recycling. viii) under appropriate conditions of flocculation MTCC Y0049 settle or sediment within 10 seconds to 55 seconds. ix) properties described above are quite stable. The biochemical properties of the improved strains, MTCC Y0045, MTCC Y0046, MTCC Y0047, MTCC Y0048 and MTCC Y0049, are that they utilise glucose, sucrose, maltose, saccharose and raffinose as carbon sources but do not grow in salicin, lactose, inositol, citrate, 2-Keto-D-glucorate, arabinose, xylose, adanitol, xylitol, sorbitol, methyl-D-glycoside, n-acetyl-glucosamine, cellobiose, trehalose and melizitose. Growth was poor when sodium nitrate, potassium nitrate or lysine were used as a sole source of nitrogen. Molasses is a by-product of sugar manufacturing process. What remains after sugar is extracted from sugar cane juice is known as molasses. This by-product still contains some sugar, concentration of which depends on the efficiency of sugar extraction. A good quality (Grade A) molasses contains a minimum of 55% (w/v) sugar. A Grade C molasses on the other hand, has about 40% (w/v) sugar. In order to achieve a desired sugar concentration in a fermer'.er. molasses is accordingly diluted with water. Since ethanolic fermentation is a process by which certain microorganisms convert sugars such as sucrose, glucose and fruc'.ose to ethyl alcohol; the sugars used in the process of the present invention can be glucose, sucrose, fructose or other reducing sugars as such or as present in molasses or a combination of these or the sugars obtained from starch and other lignocellulosic material. Since conventional yeast strains prefer a temperature of around 30°C for fermentation, the fermenters are provided with a cooling device to maintain the temperature. Rise in temperature due to breakdown of cooling device, power failure, heat generated due to metabolic activities of yeast or due to any other cause results in inefficient or complete stoppage of fermentation. As a result, valuable resources are wasted. This type of problem can occur throughout the year specially in summer when ambient temperature may go well over 40°C in many parts of the country. The strains developed in the present invention are capable of fermentation at normal temperature as well as higher temperature. Thus, fermentation can be best effected at a temperature as high as 38°C, by design, or due to accidental breakdown of the cooling system for certain period during fermentation. The strains also ferment sugars at temperatures lower than 38°C with equal efficiency. Accordingly the present invention provides an improved process for the production of alcohol using improved strain of Saccharomyces cereviceae which comprises growing thermotolerant flocculent strain of Saccharomyces having characteristic such as herein described , in liquid YEPD ( Yeastextractpeptone Dextrose) medium , inoculating the liquid culture of said Saccharomyces strain to molasses medium containing 20% total sugar , culturing at a temperature ranging 15-40 ° C for 16 -96 hrs , adding inoculum of obtained culture in fresh molasses medium at 38 ° C for 48 hrs at 150 rpm .recovering the alcohol produced with yeild percentage ranging between 7- It could be observed from the description given above that the process of the present invention results in a series of new strains which have improved characteristics which can be utilised for the production of high percentage of ethanol. The process of the present invention is illustrated in the examples given below which should not, however, be construed to limit the scope of the present invention. Example 1 A diploid homothallic strain of Saccharomyces cerevisiae MTCC Y0001 was grown in YEPD (yeast extract, peptone, dextrose, distilled water) medium at 30°C for 24 hours and cells were harvested. The cells were treated with lithium acetate and DNA mediated transformation was carried out as described by Kaiser, et al. (1994, Methods in Yeast Genetics, CSHL, Press, USA). Transformants were selected on YEPD medium containing an antibiotic G418. One transformant was selected and designated as T2. This strain T2 was spread over sporulation medium and incubated at 25°C for 3 days. The asci were harvested, treated with zymolyase and spores (four per ascus) were separated with the help of a micromanipulator. Individual spores were checked for G418 resistance, ploidy and mating types. Cells derived from two spores having antibiotic resistance were selected and were designated as T2-2A and T2-2C. They are haploid and are of a ar^ slpha mating type, respectively. Strain T2-2C was mutagerised with a chemical mutagen ethyl methane sulfonate and the mutagenised population was subjected to enrichment and selection for fermentation ability at high temperature in YEPS medium (Yeast extract, Peptone, Sucrose). Fermentation, as determined by loss of weight of the medium in the flasks (due to generation of carbon dioxide which is lost), was monitored at regular intervals. Once fermentation was complete, 3 ml of this culture was added to 30ml of fresh YEPS and fermentation was carried out at 37°C as before. This enrichment for thermotolerant mutant was repeated a totai of six limes. After final round of fermentation, ceils were purified to single colonies on YE?D and were individually tested for their fermentation ability and viability at high temperature (38°C). The resulting strain showing good viability and ethanol production at this temperature was selected and designated as MTCC Y0045. The improved strain was inoculated in YEPD medium and incubated at 30°C with shaking for 24 hrs. Sample of this culture was added to YEPS containing 20% sugar. The flasks were incubated with shaking at 38°C for 48 hours. Samples were withdrawn at regular intervals and sugar contents and alcohol produced were checked by standard anthrone and potassium dichromate methods respectively. Example 2 A strain of Saccharomyces cerevisiae MTCC Y0001 was grown in YEPD (yeast extract, peptone, dextrose, distilled water) medium at 30°C for 24 hours and cells were harvested. The cells were treated with lithium acetate and DNA mediated transformation was carried out as described by Kaiser, et al. (1994, Methods in Yeast Genetics, CSHL, Press, USA). Transformants were selected on YEPD medium containing an antibiotic G418. One transformant was selected and designated as T2. This strain T2 was spread over sporulation medium and incubated at 25°C for 3 days. The asci were harvested, treated with zymolyase and spores (four per ascus) were separated with the help of a micromanipulator. Individual spores were checked for G418 resistance, ploidy and mating types. Cells derived from two spores having antibiotic resistance were selected and were designated as T2-2A and T2-2C. They are haploid and are of a and alpha mating type respectively. Strain T2-2C was mutagenised with a chemical mutagen ethyl methane sulfonate and the mutagenised population was subjected to enrichment and selection for fermentation ability at high temperature in YEPS medium (Yeast extract, Peptone, Sucrose). Fermentation, as determined by loss of weight of the medium in the flasks (due to generation of carbon dioxide which is lost), was monitored at regular intervals. Once fermentation was complete, 3 ml of this culture was added to 30ml of fresh YEPS and fermentation was carried out at 37°C as before. This enrichment for thermotolerant mutant was repeated a total of six times. After final round of fermentation, cells were purified to single colonies on YEPD and were individually tested for their fermentation ability and viability at high temperature (38°C). The resulting strain showing good viability and ethanol production at this temperature was selected and designated as MTCC V0045. Liquid (YEPD) culture of the improved strain MTCC Y0045 was inoculated to molasses medium containing 20% total sugar and incubated with shaking at 38°C for 16 hours. This inoculum was added to a fresh molasses medium containing 20% total sugar and incubated at 38°C. Incubation was carried out with shaking at 150 rpm for 48 hours. Samples were withdrawn at regular intervals and alcohol produced was estimated. Sugar contents and alcohol produced were determined by standard anthrone and potassium dichromate methods respectively Example 3 A strain of Saccharomyces cerevisae T2-2C as described in Example 1 was grown in YEPD for 20 hours at 30°C. The culture was treated with ethidium bromide and from the treated population of cells, a respiratory deficient mutant, designated as T2-2C-0 was selected. This strain is haploid, can not grow on glycerol and is resistant to an antibiotic G418. This strain was crossed with another strain YNN217 (haploid, can grow on glycerol and is sensitive to G418; obtained from Prof. Ronald Davis, Stanford University, USA). From this cross, diploids, which can grow on glycerol and resistant to G418, were selected. A diploid strain was grown on sporulation medium for 3 days at 30°C. Cells were harvested and treated with zymolyase, spores (four spores per ascus) were separated with a micromanipulator and individual spores were tested for G418 resistance and respiratory deficiency. Haploid G418 sensitive cells without respiratory deficiency were selected and grown in YEPD at 30°C. One of these was back-crossed with T2-2C-0. In a back- cross, diploid cells are sporulated to produce haploid spores and the haploid spores (with proper mating type) are crossed with one of the two original parents which took part to produce diploid cells in the first place. Such back-crossing was carried out a total of six times. This ensured that final progeny has most of the genetic material of strain T2-2C and incorporate desired ho character from the other parent. The strain finally selected is designated as 38-2C. This strain is a stable haploid and is sensitive to G418. Strain 38-2C was mutagenised with a chemical mutagen ethyl methane sulfonate and the mutagenised population was subjected to enrichment and selection for fermentation ability at high temperature in YEPS medium (Yeast extract, Peptone, sucrose). Growth and fermentation, as determined by loss of weight of the flasks, was moniiored at regular intervals. Once fermentation was complete, 3 ml of this culture was added to 30ml of fresh YEPS and fermentation was carried out at 37°C as before. This enrichment for thermotolerant mutant was repeated a total of six times. After final round of fermentation, cells were purified to single colonies on YEPD and were individually tested for their fermentation ability and viability at high temperature (38°C). The resulting strain showing good viability and alcohol production at this temperature was selected and designated as MTCC Y0046. The improved strain was inoculated in YEPD medium and incubated at 30°C with shaking for 24 hrs. Sample of this culture was added to YEPS containing 20% sugar. The flasks were incubated with shaking at 38°C for 48 hours. Samples were withdrawn at regular intervals and sugar contents and alcohol produced were checked by standard anthrone and potassium dichromate methods respectively. Example 4 A strain of Saccharomyces cerevisae T2-2C as described in Example 1 was grown in YEPD for 20 hours at 30°C. The culture was treated with ethidium bromide and from the treated population of cells, a respiratory deficient mutant, designated at T2-2C-0 was selected. This strain is haploid, can not grow on glycerol and is resistant to an antibiotic G418. This strain was crossed with another strain YNN217 (haploid, can grow on glycerol and is sensitive to G418; obtained from Prof. Ronald Davis, Stanford University, USA). From this cross, diploids, which can grow on glycerol and resistant to G418, were selected. A diploid strain was grown on sporulation medium for 3 days at 30°C. Cells were harvested and treated with zymolyase, spores (four spores per ascus) were separated with a micromanipuiaior and individual spores were tested for G418 resistance and respiratory deficiency. Haploid G418 sensitive cells without respiratory deficiency were selected and grown in YEPD at 30°C. One of these was back-crossed with T2-2C-0. In a back-cross, diploid cells are sporulated to produce haploid spores and the haploid spores (with proper mating type) are crossed with one of the two original parents which took part to produce diploid cells in the first place. Such back-crossing was carried out a total of six times. This ensured that final progeny has most of the genetic material of strain T2-2C and incorporate desired ho character from the other parent. The strain finally selected is designated as 38-2C. This strain is a stable haploid and is sensitive to G418. Strain 38-2C was mutagenised with a chemical mutagen ethyl methane sulfonate and the mutagenised population was subjected to enrichment and selection for fermentation ability at high temperature in YEPS medium (Yeast extract, Peptone, sucrose). Growth and fermentation, as determined by loss of weight of the flasks, was monitored at regular intervals. Once fermentation was complete, 3 ml of this culture was added to 30ml of fresh YEPS and fermentation was carried out at 37°C as before. This enrichment for thermotolerant mutant was repeated a total of six times. After final round of fermentation, cells were purified to single colonies on YEPD and were individually tested for their fermentation ability and viability at high temperature (38°C). The resulting strain showing good viability and alcohol production at this temperature was selected. Liquid (YEPD) culture of the improved strain MTCC Y0046 was inoculated to molasses medium containing 20% total sugar and a part of the culture was incubated with shaking at 38°C for 16 hours, the other at of 30CC for the same period. Inoculum made at 38°C was added to a fresh molasses medium containing 20% total sugar and incubated at 38°C, the other inoculum grown at 30°C was added to molasses (20% total sugar) and incubated at 30° C. Incubation was carried out with shaking at 150 rpm for 48 hours. Samples were withdrawn at regular intervals. Alcohol production at both temperatures were monitored. Sugar contents and alcohol produced were determined by standard anthrone and potassium dichromate methods respectively. Example 5 A strain of Saccharomyces cerevisiae MTCC Y0001 was grown in YEPD at 30°C for 24 hours. The cells were transferred to a sporulation medium and incubated for 3 days. Cells from sporulation medium were treated with zymolyase. Individual cells were taken out with a micromanipulator and arranged in rows. Cell of another strain MTCC Y0046 were picked one at a time with a micromanipulator and placed next to a spore (already arranged in rows) touching it. Mating between the sp^rc? and cello were followed microscopically. Diploids were selected and they were sporulated and spores were separated as described above. The spores were allowed to grow on YEPD and cells which remained haploid were heterothallic and were discarded. Some of the spores produced cells which became diploid by switching mating type by some of the cells and mating with the cells having opposite mating type. The resulting strain was selected and designated as MTCC Y0048. Liquid (YEPD) culture of the improved strain MTCC Y0048 was inoculated to molasses medium containing 18% total sugar and incubated with shaking at 380C for ib hours. This inoculum was added to a fresh molasses medium containing 18% total sugar and incubated at 38°C. Incubation was carried out with shaking at 150 rpm for 48 hours. Progress of fermentation was monitored in terms of weight loss (due to liberation of carbon dioxide) at regular intervals. Alcohol production was calculated from this weight loss. Example 6 A strain of Saccharomyces cerevisiae MTCC Y0046 was grown in YEPD. Another strain MTCC Y0002 of opposite mating type was also grown in YEPD. These two cells are mixed. The mixture was then spread on complete Yeast Nitrogen Base Glucose medium and incubated at 25°C. Cells from the above plates are harvested and then spread on Yeast Nitrogen Base Glycerol medium containing an antibiotic. Cells growing on this medium are considered to incorporate desired properties from both the parents since neither of the parents can grow on this medium. Cells from the selective plates were purified to single colonies. Pure cultures were then inoculated in 2', til YEPD medium in 24 well microtitre plates. The plates were incubated at 38°C with shaking at 150 rpm for 24 hours. Flocculation was observed as accumulation of majority of cells as small granules at the bottom of the wells. This flocculation can also be visualised in flasks containing liquid medium like YEPD, YEPS, buffered YNB glucose, molasses etc. The diploid cells were grown in YEPD and spread on sporulation medium and incubated for 3 days at 30°C. Spores were separated with a micromanipulator and were allowed to grow into haploid cells. The spore clones were tested for flocculation as described above. From among these clones strain TF2-9C was selected and was crossed with MTCC Y0046. A diploid strain originating from the cross was selected and designated as TF5. Another back cross of MTCC Y0046 with spores of strain TF5 was performed to generate diploids and one such strain T6 having properties of flocculation, thermotolerance and fermentation ability at high temperature was selected, this T6 strain was sporulated and spores were individually tested for the presence of desired properties. The resulting strain was found to be flocculent, thermotolerant and is efficient in fermentation at high temperature and designated as MTCC Y0049. Liquid (YEPD) culture of the improved strain MTCC Y0049 was inoculated to molasses medium containing 18.2% total sugar and a part of the culture was incubated with shaking at 38°C for 16 hours, the other at 30°C for the same period. Inoculum made at 38°C was added to a fresh molasses medium containing 18.2% total sugar and incubated at 38°C, the other inoculum grown at 30°C was added to molasses and incubated at 30°C. Distinguishing features as compared with prior art: Advantages of the Invention 1. A process for the preparation of improved thermotolerant and flocculent strain of Saccharomyces cerevisiae useful for the fermentative production of ethanol wherein the improved strains of yeast produced are thermotolerant and are capable of fermentation at higher temperature. 2. Viability of the improved strains produced in high temperature fermentation is more than conventional yeast strains. As a result, in case of rise of temperature in the fermenter due to high ambient temperature, power failure, break-down of cooling device etc, the process will continue to completion and valuable resources will not go waste as it happens with conventional yeast. 3. One improved strain of yeast produced has additional property of flocculation and as a result, majority of the cells sediment at the bottom when agitation is stopped. 4. In addition, the improved yeast strains produced are more osmotolerant, more ethanol tolerant and as a result, produce higher level of alcohol at normal as well as at higher temperature compared to conventional yeast strains without any loss of their ability for conversion of sugar to alcohol. 5. Because of above said properties of the improved strains, higher initial sugar concentration can be used for fermentation. Consequently, for a given capacity plant, alcohol output may be higher depending on the conditions used. 6. Because of high alcohol content in the wash, which is achieved by high gravity fermentation, there is substantial reduction in steam consumption in the recovery process. Net saving of steam in distillation process can be between 0.8 kg to 1.2kg per litre of alcohol distilled. 7. Consequent to high gravity fermentation, there is a net reduction in effluent volume. This may result in a more compact effluent treatment plant. 8. The improved strains are genetically marked and because of this the strains are easily identifiable. We claim: 1. A process for the preparation of improved thermotolerant and flocculent strain of Saccharomyces cerevisiae useful for the fermentative production of ethanol by a process which comprises: a) preparing a stable haploid of Saccharomyces cerevisiae by HO gene disruption, b) characterized in treating the haploid derivatives obtained in the above step with mutagen belonging to group comprising MNNG (N-methyl-N5-nitro-N-nitrosoguanidine), EMS (ethyl methanesulfonate), UV, (ultraviolet light) and related agents. c) subjecting the mutant population of cells obtained in the above step, to high temperature fermentation wherein the temperature ranges from 15-40 degree centigrade; d) reusing the surviving cells obtained in the above step, in subsequent high temperature fermentation, repeating the step few more times to enrich mutant population, e) introducing flocculation property in some of the strains obtained in steps described above, f) carrying out fermentation in a temperature range of 15 °C to 40°C. 2. A process as claimed in claim 1, wherein the fermentation is carried in a rich medium containing fermentable sugars wherein the sugars used are glucose, sucrose, maltose, saccharose and raffinose as carbon sources. 3. A process as claimed in claim numbers 1-2, wherein the fermentation is carried out in a temperature range of 15°C to 40°C for a time period of about 16 to 96 hours. 4. A process as claimed in claim numbers 1-3, wherein the ethanol produced is in the range of 7% to 12%v/v. 5. A process as claimed in claims 1-4, wherein the yeast strain used is a haploid. 6. A process as claimed in claims 1-5, wherein the yeast strain used is a diploid. 7. Recombinant thermotolerant strain of Saccharomyces cerevisiae characterized in producing 7% to 12% v/v ethanol at a temperature range between 28°C and 38°C.

Full Text

1 The invention relates to a process for the preparation of improved
thermotolerant focculent Saccharomyces. This invention
particularly relates to an improved yeast strain of Saccharomyces cerevisiae useful for the preparation of ethanol by the fermentation of sugars. The ethanol so prepared is useful both for potable and industrial purposes. /
It is well known that ethanol or ethyl alcohol (C2H5OH) is produced by both fermentation and synthetic methods. Fermentation techniques for ethanol production developed during the early part of this century were supplemented by synthetic processes based on crude petroleum, as oil was much cheaper and abundantly available. However, of late, it is realised that petroleum oil reserve is not going to last long and fermentative production of ethanol has again picked up, using various kinds of renewable ferrnemabie subsiraics be il (i) sugar (from sugar-cane, sugar beet, fruit) which may be converted to ethanol directly; (ii) starch (from grain, root crops) which are first hydrolysed to fermentable sugars by enzymes; and (iii) cellulose (from wood, agricultural wastes, etc.) which are converted to sugars. (Biotechnology: Economic and Social Aspects :- Issues for Developing Countries. Eds. : E.J. Da Silva, C.Ratledge and A. Sesson; Cambridge University Press, p24 1992).
Ethanol production by fermentation is based mainly on yeasts, and for large scale fuel production these are generally of the genus Saccharomyces.
The distillers all over the world would like to have a fermentation process which can run at a temperature range between 28°C and 38°C and gives high percentage of alcohol in the broth without sacrificing fermentation efficiency. The primary benefits of such an operation are :
a) potential to increase the productivity (wherein productivity is defined
as amount of alcohol produced per unit fermcnter vol./hr).
b) fermenters may be operated at normal as well as higher temperature
(28°C to 38°C) by design or caused by accidental failure of cooling
device or electrical break down in the plant.
c) saving in energy/steam requirement during distillation; energy being
the major variable cost in distillery operation.
d) reduction in the volume of effluent.
e) in case of flocculent yeast strains, fermentation may be done by
recycling of cells, in a continuous mode or by conventional batch
rnnrji? Rer.ause of their flocculent nature, cells are easily separated
from the fermented broth and in a recycling mode or in a continuous
mode less sugar is expected to be required for building up of biomass.
In conventional method of ethanol production, initial concentration of sugar in the broth is kept between 14 to 16 percent. Sugar concentrations higher than this is detrimental to growth of yeast strains used in conventional process and fermentation is thus affected. After completion of fermentation, 6.5 to 8 percent alcohol is obtained in the wash. Alcohol is then recovered by distillation using steam.
In order to achieve higher ethanol level in the wash, initial sugar concentration in the broth should also be higher, thereby increasing the osmolarity of the medium which is detrimental to the growth and fermentation of the conventional yeast strains. Conventional strains are also sensitive to
high level of ethanol in the broth and they do not have flocculent characteristic.
One of the drawbacks in conventional yeast strains is their sensitivity to high temperature. High temperature in the fermenter is generated by ambient heat as well as metabolic activities of yeast strains. This necessitates efficient cooling device which may be expensive. In case of power failure or accidental break down of cooling device, fermentation does not get completed resulting in wastage of material and other resources.
Moreover, conventional yeast strains fail to grow and become inactive at high sugar concentration during fermentation process. At the same time unless high sugar concentration is used in the fermentation process, it is not possible to obtain increased level of ethanol. Concurrent with this problem conventional yeasts will not be effective in the fermentation, if ethanol concentration is increased in the broth. The cumulative effects of these problems in the conventional process of production of ethanol leads to low level of ethanol in the wash and consequently, consumption of steam per litre of ethanol distilled is high. Therefore, the processes are not efficient and also not economical. Moreover, separation of conventional yeast strains from fermented broth is not easy and may not be very efficient in a cell-recycling or continuous mode of operation.
Realising the need to have strains possessing various desirable properties and useful for high density fermentation and in a wide range of operating temperatures we continued our research with the objective of developing thermotolerant strains of yeast which, in addition to tolerating high initial sugar concentration, i.e. having osmotolerant characteristic, could
Saccharomyces particularly species cerevisiae which are useful for fermentation pr^s^s for the preparation of ethanol.
Another objective of the present invention is to introduce the flocculation characteristics in some of the said thermotolerant strains which are useful for the preparation of ethanol.
Another objective of the present invention is to produce thermotolerant strains of the genus Saccharomyces species - cerevisiae by using the genetic engineering, mutation, enrichment and repeated selection processes.
Another objective of the present invention is to introduce the flocculent characteristics in the thermotolerant strains of the genus- Saccharomyces species - cerevisiae by genetic hybridisation or cytoduction followed by selection.
By thermotolerant it is meant that the strain should be able to efficiently ferment sugars to ethanol at a higher temperature (>35°C) than that is optimal for normal strains (28°C to 32°C), it should also retain good viability at the end of fermentation, so the cells can be reused in subsequent rounds of fermentation. One common process employed so far to obtain thermotolerant strains of yeast is to screen for such strains in the natural environment (D'Amore T et al., 1989, Enzyme Microb Technol 11: 411-416; Banat IM & Marchant R, 1995, World J. Microbiol Biotechnol 11: 304-306; Banat IM et al, 1992, World J. Microbiol Biotechnol 8: 259-263; Hacking AJ et al., 1984, Appl Microbiol. Biotech. 19: 361-363; Kida et al, 1992, J. Ferment & Bioeng. 74: 169-173; Laluce C et al, 1991, Biotechnol Bioeng 37: 528-536; Rosa MF et al, 1987, Biotech Lett. 9: 441-444). Though this process is simple, it can not obviously be used to improve pre-existing strains that may have
many other desired features such as high osmotolerance, high ethanol tolerance and faster fermentation rate. One r^tbod often used to combine thermotolerance with other good properties is by protoplast fusion of strains having such properties (Kida et al. ,1992, J. Ferment & Bioeng. 74: 169-173; Seki T et al., 1983, Biotechnol Lett. 5: 351-356; Stewart GG et al., 1988, US patent No. 4,772,556). However, the disadvantage here is that unwanted features from the parent strains will also come into the hybrid strain and there is no method by which those can be readily removed from the hybrid.
Thus the present invention relates to a process for the preparation of thermotolerant strains of yeast which obviates the drawbacks detailed above and thermotolerant mutants can be obtained starting from existing diploid homotnailic strains, and the mermotoierance property so obtained can be combined with good properties from other strains. The strains are prepared such that any undesirable features introduced inadvertently can also be readily removed.
Distillery strains are usually diploid (having two sets of genes) or polyploid (having more than two sets of genes) and may be homothallic, i.e. haploid cells derived from spores can become diploid by mating with opposite mating-type (which originate in the population by switching over of mating-type). It is difficult to isolate mutants from diploid or polyploid strains since most of the mutations are recessive. That is, in a cell where normal gene and recessive mutant gene are present together, the former will mask the expression of the latter. It is, therefore, essential to generate stable haploid cells for generating mutant strains. Once desired mutants are obtained they
can be further manipulated for construction of other strains with novel characteristics.
In general, desirable genetic properties of two strains or more may be brought together, for creating novel straias by many ways such as, mating, (also known as crossing, genetic hybridisation), cytoduction, protoplast fusion as well as by genetic engineering and expression of the cloned gene(s). In doing this manipulations in the laboratory, the strains need to be properly marked and an innovative method has to be developed for the production of improved and novel strains.
Accordingly, the present invention provides a process for the preparation of improved thermotolerant flocculent strains of Saccharomyces, which comprises
a) growing a diploid homothallic strain of Saccharomyces, having
features such as osmotolerance and ethanol tolerance, in a conventional
medium, mutating the homothallic gene of the said strain by known
methods, sporulating the resulting diploid and separating the spores to
get stable haploids,
b) treating the said haploids with conventional mutagen by known
methods, and subjecting them to high temperature fermentation
repeatedly, to get stable thermotolerant strains retaining all other
properties of the parent strain such as osmotolerance and ethanol
tolerance,
c) growing a haploid strain having flocculation property using
conventional medium,
d) mating cells obtained in step (b) and (c) by known methods,
e) isolating the resultant diploid cells by known methods, and checking
for ploidy, prototrophy, antibiotic resistance and flocculation,
f) sporulating the hybrid cells obtained in step (e) by known methods and
separating individual spores from ascus using conventional methods,
g) mating haploid cells obtained in step (f) with haploid cells of opposite
mating type obtained in step (b) to get diploid cells having
thermotolerant and flocculent characters,
h) sporulating the cells obtained in step (g) and isolating the spores by known methods, and testing the resultant haploid cells for thermotnlerance; flocculation and other desirable properties.
i) mating haploid cells obtained in step (h) with cells obtained in step (b), sporulating the resultant diploid and checking the resulting haploid cells for thermotolerance, flocculation and other desirable properties to get improved stable strains of thermotolerant and flocculent Saccharomyces useful for improved fermentation of fermentable sugars at high temperature.
The homothallic strain of Saccharomyces cerevisiae used in step (a), deposited at Microbial Type Culture Collection and designated as MTCC Y0001, has the following properties: It is homothallic, diploid, prototrophic, osmotolerant, non-flocculent, and makes 10 to 12% (v/v) alcohol at 30°C, which is a subject matter of a copending patent application no. 748/DEL/93. This has been deposited at NCYC and has the accession no. NCYC 2646.

The flocculent strain used in step (c), deposited at Microbial Type Culture Collection and designated as MTCC Y0002, and also having ATCC accession no. ATTC 90506, has the following properties: It is heterothallic, haploid, auxotrophic, flocculent, and produces 2 to 3 % (v/v) alcohol at 30°C.
In a preferred embodiment the growth of MTCC Y0001 may be effected in a known medium such as YEPD (Yeast extract, Peptone, Dextrose) at a temperature in the range of 15°C to 35°C for a period of 1 to 10 days.
In another preferred embodiment the HO gene in MTCC Y0001 may be mutated by conventional mutagenesis or by targeted gene disruption or by genetic hybridisation method.
In yet another preferred embodiment the medium used for sporulating the diplcid strains such as Y0001 may consist of Agar, Yeast extract, Dextrose, Potassium acetate, and distilled water and the lytic enzyme used may be selected from lyticase, glusulase or zymolyase.
In yet another preferred embodiment the mutagen used in step (b) may be selected from conventional chemical mutagens such as N-methyl-N'-nitro-N-nitrosoguanidine and ethyl methanesulfonate, or from ultraviolet light radiation, gamma radiation or similar agents.
In yet another preferred embodiment fermentation may be carried out in a medium such as YEPD, YEPS (Yeast extract, Peptone, Sucrose), molasses or other media containing fermentable sugar at a temperature in the range of 15°Cto40°C.
In yet another preferred embodiment the thermotolerant strains obtained in step (b) can be used as such for the fermentative production ^f ethanol.
In yet another preferred embodiment the haploid strain MTCC Y0002 (ATCC 90506) used in step (c) may be selected from a haploid strain as such or haploid obtained from diploid strains. By way of example, other properly marked strains may be used.
In yet another preferred embodiment the haploid strain in step (c) is grown in a known medium such as YEPD (Yeast extract, Peptone, Dextrose) at a temperature in the range of 15°C to 35°C for a period of 1 to 10 days.
In yet another preferred embodiment the mating of cells obtained in steps (b) and (c) may be effected by cell-cell pairing or by mixing and plating on a suitable medium and incubating at a temperature in the range of 15°C to 37°C for a period in the range of 1 to 10 days. The medium used in step (e) may be selected from SD Medium (yeast nitrogen base, dextrose, agar and distilled water), YPD (Yeast extract, Peptone, Dextrose, Agar and distilled water) or YPG (Yeast extract, Peptone, Glycerol, Agar and distilled water).
hi yet another preferred embodiment the medium used in step (e) for isolating diploids may be a conventional medium such as SDG (Yeast nitrogen base, Dextrose, Glycerol, Agar and distilled water) fortified with broad range antibiotic such as geniticin, oligomycin, chloramphenicol and the likes.
In yet another preferred embodiment the different strains may separated by conver^cra! methods like streaking or dilution plating or by micromanipulation.
In yet another preferred embodiment the improved thermotolerant flocculent strains may be either a haploid or diploid.
The details of the steps of the process of the present invention are as follows:
The parental strain designated as MTCC Y0001 is homothallic, diploid, prototrophic, osmotolerant, non-flocculent, and makes 10 to 12% v/v alcohol at 30°C. It can sporulate and produce haploid spores of a and alpha mating types. These spores spontaneously become diploids by switching mating types, and by subsequent hybridisation of cells of opposite mating types. To obtain stable haploid derivatives, the HO gene ( haploid gene ) of this strain, responsible for homothallism or persistent diploidy, was replaced by a mutated version (ho::neo, where it is disrupted by neo gene conferring resistance to antibiotic G418; van Zyl et al, 1993, Current Genetics 23: 290-294) by DNA mediated transformation. The transformants were selected on G418 containing medium. One of the resulting strains designated as T2 (Transformant no. 2) was sporulated on appropriate medium and spores (four per ascus) were separated with the help of a micromanipulator. Two of these four spores were sensitive to the antibiotic G418 and became diploid. The other two spores which were resistant to the said antibiotic and remained haploid were designated as T2-2A and T2-2C. While T2-2A is of a mating type, T2-2C is of alpha mating type. From these two strains respiratory deficient mutants which can not grow on glycerol were selected and designated
as T2-2A-0 and T2-2C-0 respectively. One of the strains, T2-2C-0, was crossed with the heterothallic strain YNN217 (Ramer et al, 1992, Proc. Natl. Acad. Sci. 89: 11589-11593; obtained from Prof. Ronald Davis, Stanford University, USA) and diploids were selected. The selective medium was such that the original two strains (T2-2C-0 and YNN217) should not survive. Diploids only incorporating features of both the parents would be able to grow. A diploid strain was sporulated and the spores were separated as stated above. Two of the resulting four spores were resistant to G418 while the other two were sensitive and carried the ho gene. A G418 sensitive haploid strain was crossed with T2-2C-0 described above to produce a diploid strain. This diploid strain was sporulated, spores were separated and analysed for desired characteristics. The baplnid strain with desired characteristics was crossed again (back crossing) with T2-2C-0 and sporulated and spores analysed. This back crossing was done a total of six times to ensure retention of all the desired properties and finally two haploid strains 38-2A and 38-2C, having the ho gene ( haploid gene) from strain YNN217, and of mating types alpha and a, respectively, were selected.
Two strains T2-2C and 38-2C described above were mutagenised with ethyl methane sulfonate and the mutagenised population was subjected to selection at high temperature fermentation in YEPS (Yeast extract, Peptone, Sucrose 18% w/v). After fermentation was complete, a portion of the cells were inoculated to fresh YEPS and fermentation was carried out as described above. This process was repeated six times and cells from last round of fermentation was plated out and individual colonies were tested for fermentation in YEPS at 38°C. Two strains designated as (MTCC Y0045) and
(MTCC Y0046) derived from T2-2C and 38-2C, respectively, were finally selected.
These two strains are stable haploid, thermotolerant, produce more ethanol at 38°C and retain higher viability at 38°C compared to the original parent strain MTCC Y0001. Genetic analysis showed that strain MTCC Y0045 is of alpha mating type and strain MTCC Y0046 belongs to a mating type. Mutations in these strains are in two different genes but both of them are recessive to wild type.
Diploid versions of these haploid thermotolerant strains were also constructed by genetic crosses. Strain MTCC Y0046 (a mating type) was crossed with a strain 38-2A (alpha mating type and non thermotolerant). The resulting diploid strains were sporulated and spore clones were analysed as stated earlier. A thermotolerant clone of alpha mating type (106-2A) was selected and mated with MTCC Y0046. The resulting diploid strain designated as MTCC Y0047 was found to have high temperature fermentation ability comparable to haploid MTCC Y0046. In another strategy, spores of MTCC Y0001 were separated individually with the help of a micromanipulator and cells of MTCC Y0046 were placed next to these spores for mating, which was monitored under a microscope. The resulting diploids were sporulated and spores from individual ascus were allowed to form colonies on appropriate medium. Some of these haploid spores which received HO allele from MTCC Y0001 will switch mating type and thus generate diploid cells in the population. Few such diploids were analysed. One strain designated as MTCC Y0048 was found to have thermotolerance

and high temperature fermentation efficiency comparable to original MTCC Y0046 haploid strain.
In order to construct further improved strains having characteristics such as osmotolerance, ethanol tolerance, flocculation and capable of fermentation at high temperature, haploid strain MTCC Y0046 was crossed with another haploid strain designated as MTCC Y0002 as described earlier. Cells of these two haploid strains were placed next to each other in pairs, incubated till colonies appeared on plates and the colonies were then streaked on non-selective medium. Single colonies were picked and checked for desirable properties on appropriate selective medium. The selective medium was designed such that the original two strains (MTCC Y0046 and MTCC Y0002) would not survive. Only hybrids creaied by transfer of desirable genetic material from both MTCC Y0046 and MTCC Y0002 would be able to grow, and there were many such hybrids. The hybrids were sporulated and the spores were then initially checked for flocculation, nutritional requirements, thermotolerance and mating type. Some of them showed good flocculation, some showed mild flocculation while others did not flocculate. One of the spore clones designated as TF2-9C having flocculent, thermotolerant, high temperature fermentation properties and is of alpha mating type was mated with MTCC Y0046. A diploid strain designated as TF5 having desired properties was chosen. The strain TF5 was sporulated and the spores were analysed and one spore clone named TF5-5B having desired characteristics was crossed with MTCC Y0046 and a diploid strain designated as TF6 was obtained.
TF6 was again sporulated and after separation of individual spores and their characterisation, a strain MTCC V0049 was selected. This strain is haploid, thermotolerant, flocculent and is efficient in fermentation at high temperature.
The improved strains have been deposited in the National Facility on Microbial Type Culture Collection and Gene Bank (MTCC) located at the Institute of Microbial Technology, a constituent laboratory of Council of Scientific and Industrial Research. They have been assigned the following accession numbers:
MTCC Y0045 haploid, thermotolerant, mutant derivative of T2-2C
MTCC Y0046 haploid, thermotolerant, mutant derivative of 3 8-2C
MTCC Y0047 diploid, therrrotolerant, derived from 106-2AxY0046
MTCC Y0048 diploid,, thermotolerant, derived from Y0046xYOOO1
MTCC Y0049 haploid, thermotolerant, flocculent derived from TF6
The improved strains listed above have the following characteristics:
(i) they grow at a temperature ranging between 15°C and 38°C in Yeast Extract Peptone Dextrose (YEPD) medium with 2% glucose,
(ii) they grow on agar plates containing molasses concentration of up to 50% and do not need any supplementation when grown in this medium,
(iii) they produce 7% to 11.6% v/v ethanol at a temperature range between 28°C and 38°C with up to 90% efficiency,
(iv) they also grow in Yeast Extract, Peptone, Dextrose (YEPD) medium in presence of up to 12% ethanol. The strains are, therefore,
osmotolerant, ethanol tolerant, thermotolerant and produce high level of alcohol at temperature between 28°C and 38°C.
v) in liquid medium, like Yeast Extract, Peptone, Dextrose or Yeast Extract Peptone Sucrose (YEPS), molasses or the like MTCC Y0049 shows flocculation.
vi) MTCC Y0047 and MTCC Y0048 sporulate and produce ascospores. vii) they retain their viability and fermentation ability for use in recycling.
viii) under appropriate conditions of flocculation MTCC Y0049 settle or sediment within 10 seconds to 55 seconds.
ix) properties described above are quite stable.
The biochemical properties of the improved strains, MTCC Y0045, MTCC Y0046, MTCC Y0047, MTCC Y0048 and MTCC Y0049, are that they utilise glucose, sucrose, maltose, saccharose and raffinose as carbon sources but do not grow in salicin, lactose, inositol, citrate, 2-Keto-D-glucorate, arabinose, xylose, adanitol, xylitol, sorbitol, methyl-D-glycoside, n-acetyl-glucosamine, cellobiose, trehalose and melizitose. Growth was poor when sodium nitrate, potassium nitrate or lysine were used as a sole source of nitrogen.
Molasses is a by-product of sugar manufacturing process. What remains after sugar is extracted from sugar cane juice is known as molasses. This by-product still contains some sugar, concentration of which depends on the efficiency of sugar extraction. A good quality (Grade A) molasses contains a minimum of 55% (w/v) sugar. A Grade C molasses on the other hand, has
about 40% (w/v) sugar. In order to achieve a desired sugar concentration in a fermer'.er. molasses is accordingly diluted with water.
Since ethanolic fermentation is a process by which certain microorganisms convert sugars such as sucrose, glucose and fruc'.ose to ethyl alcohol; the sugars used in the process of the present invention can be glucose, sucrose, fructose or other reducing sugars as such or as present in molasses or a combination of these or the sugars obtained from starch and other lignocellulosic material.
Since conventional yeast strains prefer a temperature of around 30°C for fermentation, the fermenters are provided with a cooling device to maintain the temperature. Rise in temperature due to breakdown of cooling device, power failure, heat generated due to metabolic activities of yeast or due to any other cause results in inefficient or complete stoppage of fermentation. As a result, valuable resources are wasted. This type of problem can occur throughout the year specially in summer when ambient temperature may go well over 40°C in many parts of the country. The strains developed in the present invention are capable of fermentation at normal temperature as well as higher temperature. Thus, fermentation can be best effected at a temperature as high as 38°C, by design, or due to accidental breakdown of the cooling system for certain period during fermentation. The strains also ferment sugars at temperatures lower than 38°C with equal efficiency.
Accordingly the present invention provides an improved process for the production of alcohol using improved strain of Saccharomyces cereviceae which comprises growing thermotolerant flocculent strain of Saccharomyces having characteristic such as herein described , in liquid YEPD (
Yeastextractpeptone Dextrose) medium , inoculating the liquid culture of said Saccharomyces strain to molasses medium containing 20% total sugar , culturing at a temperature ranging 15-40 ° C for 16 -96 hrs , adding inoculum of obtained culture in fresh molasses medium at 38 ° C for 48 hrs at 150 rpm .recovering the alcohol produced with yeild percentage ranging between 7-
It could be observed from the description given above that the process of the present invention results in a series of new strains which have improved characteristics which can be utilised for the production of high percentage of ethanol.
The process of the present invention is illustrated in the examples given below which should not, however, be construed to limit the scope of the present invention.
Example 1
A diploid homothallic strain of Saccharomyces cerevisiae MTCC Y0001 was grown in YEPD (yeast extract, peptone, dextrose, distilled water) medium at 30°C for 24 hours and cells were harvested. The cells were treated with lithium acetate and DNA mediated transformation was carried out as described by Kaiser, et al. (1994, Methods in Yeast Genetics, CSHL, Press, USA). Transformants were selected on YEPD medium containing an antibiotic G418. One transformant was selected and designated as T2. This strain T2 was spread over sporulation medium and incubated at 25°C for 3 days. The asci were harvested, treated with zymolyase and spores (four per ascus) were separated with the help of a micromanipulator. Individual spores were checked for G418 resistance, ploidy and mating types. Cells derived
from two spores having antibiotic resistance were selected and were designated as T2-2A and T2-2C. They are haploid and are of a ar^ slpha mating type, respectively.
Strain T2-2C was mutagerised with a chemical mutagen ethyl methane sulfonate and the mutagenised population was subjected to enrichment and selection for fermentation ability at high temperature in YEPS medium (Yeast extract, Peptone, Sucrose). Fermentation, as determined by loss of weight of the medium in the flasks (due to generation of carbon dioxide which is lost), was monitored at regular intervals. Once fermentation was complete, 3 ml of this culture was added to 30ml of fresh YEPS and fermentation was carried out at 37°C as before. This enrichment for thermotolerant mutant was repeated a totai of six limes. After final round of fermentation, ceils were purified to single colonies on YE?D and were individually tested for their fermentation ability and viability at high temperature (38°C). The resulting strain showing good viability and ethanol production at this temperature was selected and designated as MTCC Y0045.
The improved strain was inoculated in YEPD medium and incubated at 30°C with shaking for 24 hrs. Sample of this culture was added to YEPS containing 20% sugar. The flasks were incubated with shaking at 38°C for 48 hours. Samples were withdrawn at regular intervals and sugar contents and alcohol produced were checked by standard anthrone and potassium dichromate methods respectively.

Example 2
A strain of Saccharomyces cerevisiae MTCC Y0001 was grown in YEPD (yeast extract, peptone, dextrose, distilled water) medium at 30°C for 24 hours and cells were harvested. The cells were treated with lithium acetate and DNA mediated transformation was carried out as described by Kaiser, et al. (1994, Methods in Yeast Genetics, CSHL, Press, USA). Transformants were selected on YEPD medium containing an antibiotic G418. One transformant was selected and designated as T2. This strain T2 was spread over sporulation medium and incubated at 25°C for 3 days. The asci were harvested, treated with zymolyase and spores (four per ascus) were separated with the help of a micromanipulator. Individual spores were checked for G418 resistance, ploidy and mating types. Cells derived from two spores having antibiotic resistance were selected and were designated as T2-2A and T2-2C. They are haploid and are of a and alpha mating type respectively.
Strain T2-2C was mutagenised with a chemical mutagen ethyl methane sulfonate and the mutagenised population was subjected to enrichment and selection for fermentation ability at high temperature in YEPS medium (Yeast extract, Peptone, Sucrose). Fermentation, as determined by loss of weight of the medium in the flasks (due to generation of carbon dioxide which is lost), was monitored at regular intervals. Once fermentation was complete, 3 ml of this culture was added to 30ml of fresh YEPS and fermentation was carried out at 37°C as before. This enrichment for thermotolerant mutant was repeated a total of six times. After final round of fermentation, cells were purified to single colonies on YEPD and were individually tested for their fermentation ability and viability at high temperature (38°C). The resulting
strain showing good viability and ethanol production at this temperature was selected and designated as MTCC V0045.
Liquid (YEPD) culture of the improved strain MTCC Y0045 was inoculated to molasses medium containing 20% total sugar and incubated with shaking at 38°C for 16 hours. This inoculum was added to a fresh molasses medium containing 20% total sugar and incubated at 38°C. Incubation was carried out with shaking at 150 rpm for 48 hours. Samples were withdrawn at regular intervals and alcohol produced was estimated. Sugar contents and alcohol produced were determined by standard anthrone and potassium dichromate methods respectively
Example 3
A strain of Saccharomyces cerevisae T2-2C as described in Example 1 was grown in YEPD for 20 hours at 30°C. The culture was treated with ethidium bromide and from the treated population of cells, a respiratory deficient mutant, designated as T2-2C-0 was selected. This strain is haploid, can not grow on glycerol and is resistant to an antibiotic G418. This strain was crossed with another strain YNN217 (haploid, can grow on glycerol and is sensitive to G418; obtained from Prof. Ronald Davis, Stanford University, USA). From this cross, diploids, which can grow on glycerol and resistant to G418, were selected. A diploid strain was grown on sporulation medium for 3 days at 30°C. Cells were harvested and treated with zymolyase, spores (four spores per ascus) were separated with a micromanipulator and individual spores were tested for G418 resistance and respiratory deficiency. Haploid G418 sensitive cells without respiratory deficiency were selected and grown in YEPD at 30°C. One of these was back-crossed with T2-2C-0. In a back-
cross, diploid cells are sporulated to produce haploid spores and the haploid spores (with proper mating type) are crossed with one of the two original parents which took part to produce diploid cells in the first place. Such back-crossing was carried out a total of six times. This ensured that final progeny has most of the genetic material of strain T2-2C and incorporate desired ho character from the other parent. The strain finally selected is designated as 38-2C. This strain is a stable haploid and is sensitive to G418.
Strain 38-2C was mutagenised with a chemical mutagen ethyl methane sulfonate and the mutagenised population was subjected to enrichment and selection for fermentation ability at high temperature in YEPS medium (Yeast extract, Peptone, sucrose). Growth and fermentation, as determined by loss of weight of the flasks, was moniiored at regular intervals. Once fermentation was complete, 3 ml of this culture was added to 30ml of fresh YEPS and fermentation was carried out at 37°C as before. This enrichment for thermotolerant mutant was repeated a total of six times. After final round of fermentation, cells were purified to single colonies on YEPD and were individually tested for their fermentation ability and viability at high temperature (38°C). The resulting strain showing good viability and alcohol production at this temperature was selected and designated as MTCC Y0046.
The improved strain was inoculated in YEPD medium and incubated at 30°C with shaking for 24 hrs. Sample of this culture was added to YEPS containing 20% sugar. The flasks were incubated with shaking at 38°C for 48 hours. Samples were withdrawn at regular intervals and sugar contents and alcohol produced were checked by standard anthrone and potassium dichromate methods respectively.
Example 4
A strain of Saccharomyces cerevisae T2-2C as described in Example 1 was grown in YEPD for 20 hours at 30°C. The culture was treated with ethidium bromide and from the treated population of cells, a respiratory deficient mutant, designated at T2-2C-0 was selected. This strain is haploid, can not grow on glycerol and is resistant to an antibiotic G418. This strain was crossed with another strain YNN217 (haploid, can grow on glycerol and is sensitive to G418; obtained from Prof. Ronald Davis, Stanford University, USA). From this cross, diploids, which can grow on glycerol and resistant to G418, were selected. A diploid strain was grown on sporulation medium for 3 days at 30°C. Cells were harvested and treated with zymolyase, spores (four spores per ascus) were separated with a micromanipuiaior and individual spores were tested for G418 resistance and respiratory deficiency. Haploid G418 sensitive cells without respiratory deficiency were selected and grown in YEPD at 30°C. One of these was back-crossed with T2-2C-0. In a back-cross, diploid cells are sporulated to produce haploid spores and the haploid spores (with proper mating type) are crossed with one of the two original parents which took part to produce diploid cells in the first place. Such back-crossing was carried out a total of six times. This ensured that final progeny has most of the genetic material of strain T2-2C and incorporate desired ho character from the other parent. The strain finally selected is designated as 38-2C. This strain is a stable haploid and is sensitive to G418.
Strain 38-2C was mutagenised with a chemical mutagen ethyl methane sulfonate and the mutagenised population was subjected to enrichment and selection for fermentation ability at high temperature in YEPS medium (Yeast
extract, Peptone, sucrose). Growth and fermentation, as determined by loss of weight of the flasks, was monitored at regular intervals. Once fermentation was complete, 3 ml of this culture was added to 30ml of fresh YEPS and fermentation was carried out at 37°C as before. This enrichment for thermotolerant mutant was repeated a total of six times. After final round of fermentation, cells were purified to single colonies on YEPD and were individually tested for their fermentation ability and viability at high temperature (38°C). The resulting strain showing good viability and alcohol production at this temperature was selected.
Liquid (YEPD) culture of the improved strain MTCC Y0046 was inoculated to molasses medium containing 20% total sugar and a part of the culture was incubated with shaking at 38°C for 16 hours, the other at of 30CC for the same period. Inoculum made at 38°C was added to a fresh molasses medium containing 20% total sugar and incubated at 38°C, the other inoculum grown at 30°C was added to molasses (20% total sugar) and incubated at 30° C. Incubation was carried out with shaking at 150 rpm for 48 hours. Samples were withdrawn at regular intervals. Alcohol production at both temperatures were monitored. Sugar contents and alcohol produced were determined by standard anthrone and potassium dichromate methods respectively.
Example 5
A strain of Saccharomyces cerevisiae MTCC Y0001 was grown in YEPD at 30°C for 24 hours. The cells were transferred to a sporulation medium and incubated for 3 days. Cells from sporulation medium were treated with zymolyase. Individual cells were taken out with a micromanipulator and arranged in rows. Cell of another strain MTCC Y0046
were picked one at a time with a micromanipulator and placed next to a spore (already arranged in rows) touching it. Mating between the sp^rc? and cello were followed microscopically. Diploids were selected and they were sporulated and spores were separated as described above. The spores were allowed to grow on YEPD and cells which remained haploid were heterothallic and were discarded. Some of the spores produced cells which became diploid by switching mating type by some of the cells and mating with the cells having opposite mating type. The resulting strain was selected and designated as MTCC Y0048.
Liquid (YEPD) culture of the improved strain MTCC Y0048 was inoculated to molasses medium containing 18% total sugar and incubated with shaking at 380C for ib hours. This inoculum was added to a fresh molasses medium containing 18% total sugar and incubated at 38°C. Incubation was carried out with shaking at 150 rpm for 48 hours. Progress of fermentation was monitored in terms of weight loss (due to liberation of carbon dioxide) at regular intervals. Alcohol production was calculated from this weight loss.
Example 6
A strain of Saccharomyces cerevisiae MTCC Y0046 was grown in YEPD. Another strain MTCC Y0002 of opposite mating type was also grown in YEPD. These two cells are mixed. The mixture was then spread on complete Yeast Nitrogen Base Glucose medium and incubated at 25°C. Cells from the above plates are harvested and then spread on Yeast Nitrogen Base Glycerol medium containing an antibiotic. Cells growing on this medium are considered to incorporate desired properties from both the parents since neither of the parents can grow on this medium. Cells from the selective

plates were purified to single colonies. Pure cultures were then inoculated in 2', til YEPD medium in 24 well microtitre plates. The plates were incubated at 38°C with shaking at 150 rpm for 24 hours. Flocculation was observed as accumulation of majority of cells as small granules at the bottom of the wells.
This flocculation can also be visualised in flasks containing liquid medium like YEPD, YEPS, buffered YNB glucose, molasses etc.
The diploid cells were grown in YEPD and spread on sporulation medium and incubated for 3 days at 30°C. Spores were separated with a micromanipulator and were allowed to grow into haploid cells. The spore clones were tested for flocculation as described above. From among these clones strain TF2-9C was selected and was crossed with MTCC Y0046. A diploid strain originating from the cross was selected and designated as TF5. Another back cross of MTCC Y0046 with spores of strain TF5 was performed to generate diploids and one such strain T6 having properties of flocculation, thermotolerance and fermentation ability at high temperature was selected, this T6 strain was sporulated and spores were individually tested for the presence of desired properties. The resulting strain was found to be flocculent, thermotolerant and is efficient in fermentation at high temperature and designated as MTCC Y0049.
Liquid (YEPD) culture of the improved strain MTCC Y0049 was inoculated to molasses medium containing 18.2% total sugar and a part of the culture was incubated with shaking at 38°C for 16 hours, the other at 30°C for the same period. Inoculum made at 38°C was added to a fresh molasses medium containing 18.2% total sugar and incubated at 38°C, the other inoculum grown at 30°C was added to molasses and incubated at 30°C.
Distinguishing features as compared with prior art:
Advantages of the Invention
1. A process for the preparation of improved thermotolerant and flocculent strain of Saccharomyces cerevisiae useful for the fermentative production of ethanol wherein the improved strains of yeast produced are thermotolerant and are capable of fermentation at higher temperature.
2. Viability of the improved strains produced in high temperature fermentation is more than conventional yeast strains. As a result, in case of rise of temperature in the fermenter due to high ambient temperature, power failure, break-down of cooling device etc, the process will continue to completion and valuable resources will not go waste as it happens with conventional yeast.
3. One improved strain of yeast produced has additional property of flocculation and as a result, majority of the cells sediment at the bottom when agitation is stopped.
4. In addition, the improved yeast strains produced are more osmotolerant, more ethanol tolerant and as a result, produce higher level of alcohol at normal as well as at higher temperature compared to conventional yeast strains without any loss of their ability for conversion of sugar to alcohol.
5. Because of above said properties of the improved strains, higher initial sugar concentration can be used for fermentation. Consequently, for a given capacity plant, alcohol output may be higher depending on the conditions used.
6. Because of high alcohol content in the wash, which is achieved by high gravity fermentation, there is substantial reduction in steam consumption in the recovery process. Net saving of steam in distillation process can be between 0.8 kg to 1.2kg per litre of alcohol distilled.
7. Consequent to high gravity fermentation, there is a net reduction in effluent volume. This may result in a more compact effluent treatment plant.
8. The improved strains are genetically marked and because of this the strains are easily identifiable.

We claim:
1. A process for the preparation of improved thermotolerant and flocculent strain of Saccharomyces cerevisiae useful for the fermentative production of ethanol by a process which comprises:
a) preparing a stable haploid of Saccharomyces cerevisiae by HO gene disruption,
b) characterized in treating the haploid derivatives obtained in the above step with mutagen belonging to group comprising MNNG (N-methyl-N5-nitro-N-nitrosoguanidine), EMS (ethyl methanesulfonate), UV, (ultraviolet light) and related agents.
c) subjecting the mutant population of cells obtained in the above step, to high temperature fermentation wherein the temperature ranges from 15-40 degree centigrade;
d) reusing the surviving cells obtained in the above step, in subsequent high temperature fermentation, repeating the step few more times to enrich mutant population,
e) introducing flocculation property in some of the strains obtained in steps described above,
f) carrying out fermentation in a temperature range of 15 °C to 40°C.

2. A process as claimed in claim 1, wherein the fermentation is carried in a rich medium containing fermentable sugars wherein the sugars used are glucose, sucrose, maltose, saccharose and raffinose as carbon sources.
3. A process as claimed in claim numbers 1-2, wherein the fermentation is carried out in a temperature range of 15°C to 40°C for a time period of about 16 to 96 hours.
4. A process as claimed in claim numbers 1-3, wherein the ethanol produced is in the range of 7% to 12%v/v.
5. A process as claimed in claims 1-4, wherein the yeast strain used is a haploid.
6. A process as claimed in claims 1-5, wherein the yeast strain used is a diploid.
7. Recombinant thermotolerant strain of Saccharomyces cerevisiae characterized in producing 7% to 12% v/v ethanol at a temperature range between 28°C and 38°C.

Documents:

570-DEL-2002-Abstract-(15-12-2008).pdf

570-DEL-2002-Abstract-(16-02-2009).pdf

570-DEL-2002-Abstract-(23-12-2008).pdf

570-del-2002-abstract.pdf

570-DEL-2002-Claims-(15-12-2008).pdf

570-DEL-2002-Claims-(16-02-2009).pdf

570-DEL-2002-Claims-(23-12-2008).pdf

570-del-2002-claims.pdf

570-del-2002-complete specification (granted).pdf

570-DEL-2002-Correspondence-Others-(15-12-2008).pdf

570-DEL-2002-Correspondence-Others-(23-12-2008).pdf

570-del-2002-correspondence-others.pdf

570-del-2002-correspondence-po.pdf

570-DEL-2002-Description (Complete)-(15-12-2008).pdf

570-DEL-2002-Description (Complete)-(23-12-2008).pdf

570-del-2002-description (complete).pdf

570-DEL-2002-Form-1-(23-12-2008).pdf

570-del-2002-form-1.pdf

570-del-2002-form-18.pdf

570-del-2002-form-2.pdf

570-del-2002-form-3.pdf

570-DEL-2002-Others-Document-(16-02-2009).pdf

« Previous Patent

Next Patent »

Patent Number

230490

Indian Patent Application Number

570/DEL/2002

PG Journal Number

13/2009

Publication Date

27-Mar-2009

Grant Date

26-Feb-2009

Date of Filing

21-May-2002

Name of Patentee

COUNCIL OF SCIENTIFIC AND INDUSTRIAL RESEARCH

Applicant Address

RAFI MARG, NEW DELHI-110001, INDIA

Inventors:

#	Inventor's Name	Inventor's Address
1	KALIANNAN GANESAN	INSITITUTE OF MICROBIAL TECHNOLOGY,SECTOR 39-A CHANDIGARH-160036, INDIA.
2	GANDHAM SATYANARAYANA PRASAD	INSITITUTE OF MICROBIAL TECHNOLOGY,SECTOR 39-A CHANDIGARH-160036, INDIA.
3	VISHVA MITRA SHARMA	INSITITUTE OF MICROBIAL TECHNOLOGY,SECTOR 39-A CHANDIGARH-160036, INDIA.
4	INDRANI GHOSH	INSITITUTE OF MICROBIAL TECHNOLOGY,SECTOR 39-A CHANDIGARH-160036, INDIA.
5	ROHINI CHOPRA	INSITITUTE OF MICROBIAL TECHNOLOGY,SECTOR 39-A CHANDIGARH-160036, INDIA.
6	TAPAN CHAKRABARATI	INSITITUTE OF MICROBIAL TECHNOLOGY,SECTOR 39-A CHANDIGARH-160036, INDIA.

PCT International Classification Number

C12N 1/16

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA