Title of Invention	" AN IMPROVED PROCESS FOR THE PRODUCTION OF ALCOHOL USING IMPROVED THERMOTOLERANT FLOCCULENT STRAINS OF YEAST SACCHAROMYCES"
Abstract	This invention relates to an improved process for the production of alcohol, characterized in fermentation of sugar at high temperature as defined herein, using improved thermotolerant flocculent strain of yeast (Saccharomyces cereviceae) having characteristic such as herein described, the said process comprises fermenting molasses medium containing 20% total sugar by inoculating liquid culture (in Yeast extract peptone Dextrose medium) of said there to tolerant strain of yeast in the said molasses medium at a temperature ranging 15-40°C for i6-96hrs, adding inoculum of obtained culture in fresh molasses medium at 38°C for 48 hrs at 150 rpm, recovering alcohol by gravity collection method with yield percentage ranging between 7-11.6% v/v. for the preparation of major variable

Title of Invention

" AN IMPROVED PROCESS FOR THE PRODUCTION OF ALCOHOL USING IMPROVED THERMOTOLERANT FLOCCULENT STRAINS OF YEAST SACCHAROMYCES"

Abstract

This invention relates to an improved process for the production of alcohol, characterized in fermentation of sugar at high temperature as defined herein, using improved thermotolerant flocculent strain of yeast (Saccharomyces cereviceae) having characteristic such as herein described, the said process comprises fermenting molasses medium containing 20% total sugar by inoculating liquid culture (in Yeast extract peptone Dextrose medium) of said there to tolerant strain of yeast in the said molasses medium at a temperature ranging 15-40°C for i6-96hrs, adding inoculum of obtained culture in fresh molasses medium at 38°C for 48 hrs at 150 rpm, recovering alcohol by gravity collection method with yield percentage ranging between 7-11.6% v/v. for the preparation of major variable

Full Text	The invention relates to an imroved process for the production of alcohol using an improved thermotolerant flocculent strains of 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 seprepared 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 vanous kinds of renewable fermentable substrates be it (i) sugar (from sugar-cane, sugar beet, fruit) which may be converted to ethanol directly, (11) 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 sagars. (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 geaecrally 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 m the broth without sacnflcing 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 fermenter 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 electncal break down in the plant. c) saving m energy/steam requirement during distillation; energy being the major vanabk cost in distiliay agemtion. d) reduction in the volume of effluent. t) in case of flocculent yeast strains, fermentation may be done by recycling of cells, m a contjasious mode or by conventional batch mode. Because of their flocc«\|Mit nature, cells are easily separated firom the fermented broth and m a recycling mode or in a continuous mode less sugar is expected to fee required for building up of biomass. In conventional method of eth^gpl production, initial concentration of sugar in the broth is kept between 14 to 16 percent. Sugar concentrations higher than this is detrimental to grovM^ of yeast strains used m conventional process and fermentation is thus affected. After completion of fermentation, 6.5 to 8 percent alcohol is obtained in #ie wash. Alcohol is then recovered by distillation using steam. In order to achieve higher emmol level in the wash, initial sugar concentration in the broth should ate) be higher, thereby increasing the osmolarity of the medium which is detim«atal 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 charactenstic. One of the drawbacks in convfntional yeast strains is their sensitivity to high temperature. High temperature m 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 devif e, fermentation does not get completed resulting m wastage of material and other resources. Moreover, conventional yeast strains fail to grow and become inactive at high sugar concentration durmg 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 ethmol. Concurrent with this problem conventional yeasts will not be effective in the fermentation, if ethanol concentration is increased m the both. 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, separatiiip of conventional yeast strains fi-om fermented broth is not easy and may mwt be very efficient in a cell-recycling or continuous mode of operation. Realising the need to have strains possessing vanous desirable properties and useful for high density fermentation and in a wide range of operatmg temperatures we continued OAir research with the objective of developing thermotolerant strains of yeast which, in addition to tolerating high initial sugar concentration, / e. hjR'iflg osmotolerant charactenstic, could survive in the higher concentration of ethanol it produces m the broth. In other words, the strain to be used should be tolerant to high concentration of ethanoi produced during the fermentation. The strains thus developed are useful in batch fermentation but can be adopted for contmuous operation as well. For use in a versatile mode of operations, ioceulent characteristic in the strain is also desirable, In order to run fermeniation m cell-recycling or continuous mode, it is desirable that the strain is of floeculent type m addition to retaining other desirable properties. Envisaged advatage of such a unique strain is that less sugar is utilised to build-up the bumass and the sugar thus saved may be converted to ethanoi. Moreover, if a strein is flocculating there is less washout of the cell dunng operation of the process. Such a strain should also give higher productivity of ethanoi. In addtion, high concentration of ethanoi in the wash results in a reduction in steam requirements for distillation. Since the strains used in conventional distillenss make 6.5 to 8% (v/v) alcohol in the wash and have a fermentation efficient of 82.9-86.90 per cent as reported by Arbatti, S.V. and Kale, V.M. in the Pioccedings of International Seminar on "Modernization of Distilleries and Breweries," (organised by All India Distillers Association, July 1989) we concentrated our research for the development of improved strains Of Saccharomyces cerevisiae which will have increased themiotolerance asri floeculent characteristic and will overcome other problems associated with the use of conventional yeast, and make 7 to 12% (v/v) or more alcofcol in the wash with a fermentation efficiency which is as good as, if not beiter than the conventional process. The main objective of the preSiBit invention is, therefore, to provide a process for the preparation of imprisred thermotolerant strains of genus - Saccharomyces particularly species cerevisiae which are useful for fermentation process for the preparationof ethanol. Another objective of the present invention is to introduce the flocculation charactenstics in seme of the said thermotolerant strains which are useful for the preparation of ethanol. Another objective of the presejaiavention is to produce thermotolerant strams of the genus Saccharomyces spicies - cerevisiae by using the genetic engmeenng, mutation, ennchment and as peated selection processes. Another objective of the presentinvention is to introduce the flocculent charactenstics m the thermotolerant strains of the genus- Saccharomyces species - cerevisiae by genetic hybridusartion or cytoduction followed by selection. By thermotolerant it is meani that the strain should be able to efficiently ferment sugars to ethanol at t 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 comman process employed so far to obtain thermotolerant strains of yeast is to swreen for such strains m the natural environment (D'Amore T et a/., 1989, Enzyme Microb Techno! 11: 411-416; Banat IM & Marchant R, 1995, World J. Microbiol Biotechnol 11: 304-306; Banat IM et al, 1992, World J. MicroMol Biotechnol 8: 259-263; Hacking AJ et a/.. 1984, Appl Microbiol. Biotech. If: 361-363; Kida et al, 1992, J. Ferment & Bioeng. lA: 169-173; Laluce C et aL,1991, BiotechnolBioeng 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 method often used to combme thermotolerance with other good proparties by protoplast fusion of strams having such properties (Kida et a/., 1912, J, Ferment & Bioeng. 74: 169-173; Seki T et a/., 1983, Biotechnol Lett 5: 351-356; Stewart GG et a/, 1988, US patent No. 4,772,556). However, the disadvantage here is that unwanted features from the parent strains will alto come into the hybrid strain and there is no method by which those can be readily removed from the hybnd. Thus the present invention relies to a process for the preparation of thermotolerant strains of yeast which obviates the drawbacks detailed above and thermotolerant mutants can be efetaiaed starting from existing diploid homothailic strains, and the thermoterance 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 homothailic, i.e. haploid cells denved from spores can become diploid by mating with opposite matmg-type (which onginate in the papulation by switching over of mating-type). It is difficult to isolate mutantsirom 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, therefase, essential to generate stable haploid cells for generating mutant strams. Once desired mutants are obtained they can be further manipulated for construction of other strains with novel charactenstics. In general, desirable genetic proerties of two strains or more may be brought together, for creating novel steams by many ways such as, mating, (also known as crossmg, genetic hybridisation), cytoduction, protoplast fusion as well as by genetic engmeering and expression of the cloned gene(s). hi doing this manipulations in the laborarory, the strains need to be properly marked and an innovative method has to be developed for the production of improved and novel strams. Accordingly, the present invention provides a process for the preparation of improved thermotolerant flocculent strains of Saccharomyces, which composes a) growing a diploid homothallic strain of Saccharomyces, having features such as osmotolerance and ethanol tolerance, in a conventional medium, mutating the homothaftic 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 thensfcetoierant strams retainmg all other properties of the parent strain such as osmotolerance and ethanol tolerance, c) growing a haploid stram 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 checkmg for ploidy, prototrophy, antibiot resistance and flocculation, f) sporulatmg the hybnd cells obttfcied in step (e) by known methods and separating individual spores from ascus using conventional methods, g) mating haploid ceils 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) sporulatmg the cells obtained a step (g) and isolating the spores by known methods, and testing the resultant haploid cells for thermotolerance, flocculation and other desirable properties, i) mating haploid cells obtained instep (h) with cells obtamed m step (b), sporulatmg the resultant diploid aad checking the resulting haploid cells for thermotolerance, flocculation and other desirable properties to get improved stable strains of thermotolerant and flocculent Saccharomyces useful for imprved fermentation of fermentable sugars at high temperature The homothallic strain of Saccharamyces cerevisiae used in step (a), deposited at Microbial Type Culture Collection and designated as MTCC YOOOl, has the following properties: 1 is homothallic, diploid, prototrophic, osmotolerant, non-flocculent, and makes 10 to 12% (v/v) alcohol at 30°C, This has been deposited at NCYC and tes the accession no. NCYC 2646. The flocculent strain used in itep (c), deposited at Microbial Type Culture Collection and designated as MTCC Y0002, and also having ATCC accession no. ATTC 90506, has the ftilowing properties: It is heterothallic, haploid, auxotrophic, flocculent, and preduces 2 to 3 % (v/v) alcohol at 30°C. In a preferred embodiment the growth of MTCC YOOOl may be effected in a known medium such as YIPD (Yeast extract. Peptone, Dextrose) at a temperature m the range of 15°C to 35°C for a penod of 1 to 10 days. In another preferred embodiin^t the HO gene in MTCC YOOOl may be mutated by conventional mutagenesfe or by targeted gene disruption or by genetic hybndisation method. In yet another preferred embocttwent the medium used for sporulating the diploid strains such as YOOOl may consist of Agar, Yeast extract. Dextrose, Potassium acetate, and distilled water and the lytic enzyme used may be selected from lyticase, glusulas or zymolyase. In yet another preferred embodiment the mutagen used m step (b) may be selected from conventional chemicsi 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 embodlEacat fermentation may be earned 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°C to 40°C. In yet another preferred embodiment the thermotolerant strains obtained in step (b) can be used as such for the fermentative production of 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 strams may be used. In yet another preferred embodiment the haploid strain in step (c) is grown in a known medium such as YHPD (Yeast extract, Peptone, Dextrose) at a temperature m the range of 15°C to35°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-celll 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 m the range of 1 to 10 days. The medium used in step (e) may be selected from SD Medium (^ast nitrogen base, dextrose, agar and distilled water), YPD (Yeast extract, feptone, Dextrose, Agar and distilled water) or YPG (Yeast extract. Peptone, Oiyeerol, Agar and distilled water). In yet another preferred embo^ment the medium used in step (e) for isolating diploids may be a convenSpnat medium such as SDG (Yeast nitrogen base, Dextrose, Glycerol, Alp- and distilled water) fortified with broad range antibiotic such as genitici% o%omycin, chloramphenicol and the likes. In yet another preferred embodiment the different strains may separated by conventional methodb like streaking or dilution plating or by micromanipulation. In yet another preferred embodimeent 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 stram designated as MTCC YOOOl is homothallic, diploid, prototrophic, osmotolerant, non-flocculent, and makes 10 to 12% v/v alcohol at 30°C. It can sporulate and poduce haploid spores of a and alpha mating types. These spores spontanususly become diploids by switching mating types, and by subsequent hytoidisation of cells of opposite matmg types. To obtain stable haploid derivsatives, the HO gene ( haploid gene ) of this strain, responsible for homothalliasn cr persistent diploidy, was replaced by a mutated version (ho:neo, where it is disrupted by neo gene confemng resistance to antibiotic G418; van Zyl et al., 1993, Current Genetics 23: 290-294) by DNA mediated transformatiin. The transformants were selected on G418 containing medium. One of the resulting strains designated as T2 (Transformant no. 2) was sporulated on apppropriate medium and spores (four per ascus) were separated with the help of a micromanipulator Two of these four spores were sensitive to the an^iotsc 0418 and became diploid. The other two spores which were resistaaat to the said antibiotic and remained haploid were designated as T2-2A an# T2-2C. While T2-2A is of a mating type, T2-2C is of alpha mating t>pe. From these two strains respiratory deficient mutants which can not grow m gij'cerol 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 ai,1992, Proc. Natl. Acad. Sci. 89: 11589-11593; obtained from Prof Ronald Davis, Stanford University, USA) and diploids were sellGted. The selective medium was such that the original two strams (T2-2C-0 aad YNN217) should not survive. Diploids only mcorporatmg 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 earned 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 charactenstics. The haploid strain with desired characteristics was crossed again (back crossing) with T2-2C-0 and sperulated and spores analysed. This back crossing was done a total of six times to ensure retention of all the desired properties and finally two hapled stems 3 8-2A and 38-2C, having the ho gene ( haploid gene) from strain YliN2I7, and of mating types alpha and a, respectively, were selected. Two strains T2-2C and 38-2C itesciibed above were mutagenised with ethyl methane sulfonate and the mutagenised population was subjected to selection at high temperature fermentSton m YEPS (Yeast extract. Peptone, Sucrose 18% w/v). After fermentatio» was complete, a portion,of the cells were inoculated to fresh YEPS and feainentation was carried out as descnbed above. This process was repeated six times and cells from last round of fermentation was plated out and mdividual colonies were tested for fermentation m YEPS at 38°C. Two stpms designated as (MTCC Y0045) and (MTCC Y0046) denved from T2-2C and 38-2C, respectively, were finally selected. These two strains are stable kaploid, thermotolerant, produce more ethanol at 38°C and retain higher vialilty at 38°C compared to the onginal parent strain MTCC YOOOl. Genetic analysis showed that strain MTCC Y0045 is of alpha mating type and stain 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 haloid thermotolerant strains were also constructed by genetic crosses. Strati MTCC Y0046 (a mating type) was crossed with a strain 38-2A (alpha nKiing type and non thermotolerant). The resulting diploid strams were sporuliiled and spore clones were analysed as stated earlier. A thermotolerant cloae of alpha mating type (106-2A) was selected and mated with MTCC Y0046. The resulting diploid strain designated as MTCC Y0047 was foui^ to have high temperature fermentation ability comparable to haploid MTCC Y0946. In another strategy, spores of MTCC YOOOl were separated individually with the help of a micromanipulator and cells of MTCC ¥0046 were placed next to these spores for mating, which was monitored undfr 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 populatiea. Few such diploids were analysed. One strain designated as MTCC Y0048 was found to have thermotolerance and high temperature fermentation elfeiency comparable to onginal MTCC Y0046 haploid strain. In order to construct further improved strains having characteristics such as osmotolerance, ethanol tolerace, flocculation and capable of fermentation at high temperature, haploid strain MTCC Y0046 was crossed with another haploid strain designated as MTCC Y0002 as descnbed earlier. Cells of these two haploid strams were placed next to each other m pairs, incubated till colonies appeared on plaHs and the colonies were then streaked on non-selective medium. Single colonies were picked and checked for desirable properties on appropnate sel#tive medium. The selective medium was designed such that the original tma strains (MTCC Y0046 and MTCC Y0002) would not survive. Only hylids created by transfer of desirable genetic matenal from both MTCC Y0006 aad 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 stfaint designated as TF5 having desired properties was chosen. The strain TFS was sporulated and the spores were analysed and one spore clone named TF5-5B having desired characteristics was crossed with MTCC Y0046 and m d^oid strain designated as TF6 was obtained. TF6 was again sporulated and 8#er separation of individual spores and their characterisation, a strain MTCC Y{X)49 was selected. This strain is haploid, thermotolerant, flocculent mi is efficient m fermentation at high temperature. The improved strams have b^i deposited m the National Facility on Microbial Type Culture Collection and Qene Bank (MTCC) located at the Institute of Microbial Technology, a constituent laboratory of Council of Scientific and Industnal Research, Tliey have been assigned the following accession numbers: MTCC Y 0045 haploid, thermotflerant, mutant denvative of T2-2C MTCC Y0046 haploid, thennol»lerant, mutant denvative of 3 8-2C MTCC Y0047 diploid, thermoteierMit, derived fi:om 106-2AxY0046 MTCC Y0048 diploid,, thermoi»lerant, derived fi-om Y0046xY0001 MTCC Y0049 haploid, thermoirferant, flocculent derived fi-om TF6 The improved strains listed abmc have the following charactenstics: (i) they grow at a temperature ranging between 15°C and 38°C m Yeast Extract Peptone Dextrose (YEPP) medium with 2% glucose, (ii) they grow on agar plates contaKling 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) ttiey also grow m Yeast Extract, Peptone, Dextrose (YEPD) medium in presence of up to 12% e^aaol. The strains are, therefore, osmotolerant, ethanol tolerant, fewiotolerant and produce high level of alcohol at temperature betwwsi 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. vu) they retain their viability and feiRientation ability for use m recycling. viu) under appropnate conditions of flocculation MTCC Y0049 settle or sediment within 10 seconds to S5 seconds. ix) properties descnbed 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, sacciiarose and raffinose as carbon sources but do not grow in salicm, lactose, inositol, citrate, 2-Keto-D-glucorate, arabinose, xylose, adanitol, xylitol, KirbMol, methyl-D-glycoside, n-acetyl-glucosamme, cellobiose, trehalose an4 melizitose. Growth was poor when sodium nitrate, potassium nitrate or lysme were used as a sole source of nitrogen. Molasses is a by-product of sugar manufactunng process. What remains after sugar is extracted from aigar 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 Gade C molasses on the other hand, has about 40% (w/v) sugar. In order to achieve a desired sugar concentration in a fermenter, molasses is accordingly diluted with water. Since ethanolic feraientatioft is a process by which certain microorganisms convert sugars such ai sucrose, glucose and fructose to ethyl alcohol; the sugars used in the process of the present invention can be glucose, sucrose, fructose or other reducing suprs as such or as present m molasses or a combination of these or the sugars obtained from starch and other lignocellulosic matenal. Since conventional yeast straiae prefer a temperature of around 30°C for fermentation, the fermenters are ptmndnd with a cooling device to maintain the temperature. Rise in temperature due to breakdown of cooling device, power failure, heat generated due to mstabolic activities of yeast or due to any other cause results in inefficient or ccjfnplete stoppage of fermentation. As a result, valuable resources are waste!. This type of problem can occur throughout the year specially m summer when ambient temperature may go well over 40°C m many parts of the country. The strains developed in the present invention are capable of fermeatation at normal temperature as well as higher temperature. Thus, fermentati«i 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 dunng fermeaction. The strains also ferment sugars at temperatures lower than 38°C with equal efficiency. Accordingly the present invention povides an improved process for the production of alcohol using improved sfrain of Saccharomyces cereviceae which composes growing thermotoloment flocculent strain of Saccharomyces having charactenstic such as herein described , in liquid YEPD Yeastextractpeptone Dextrose) medium, moculating the liquid culture of said Saccharomyces strain to molasses medium containing 20% total sugar , cultunng at a temperature ranging 15-40° C for 16 -96 hrs , addmg moculum of obtained culture m fresh molasses a\|edi»m at 38 ° C for 48 hrs at 150 rpm ,recovenng the alcohol produced with yeild percentage ranging between 7-11.6%v/v. It could be observed from the descnpfei 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 ; 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 YOOOl was grown in YEPD (yeast extract, peptone, dextrose, distilled water) medium at 30°C for 24 hours and celbwfflse harvested. The cells were treated with lithium acetate and DNA mediafted transformation was carried out as descnbed by Kaiser, et al (1994, Method in Yeast Genetics, CSHL, Press, USA). Transformants were selected on YEPD medium containing an antibiotic G418. One transformant was sdected and designated as T2. This stram 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 ofa. nacromanipulator. Individual spores were checked for G418 resistance, phidy and mating types. Cells denved from two spores having antibiotic instance 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 mutagenisedwith a chemical mutagen ethyl methane sulfonate and the mutagenised population was subjected to ennchment and selection for fermentation ability at high temperature m YEPS medium (Yeast extract, Peptone, Sucrose). Fermentialsn, as determmed 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 fr«ib YEPS and fermentation was earned out at 37°C as before. This enri4tt«ait for thermotoierant mutant was repeated a total of six times. After fteai round of fermentation, cells were punfied to single colonies on YEPD and were individually tested for their fermentation ability and viability at Ifeh temperature (38°C). The resulting strain showing good viability and etNinol production at this temperature was selected and designated as MTCC Y0W5. The improved strain was inoci4iat©d in YEPD medium and incubated at 30°C with shaking for 24 hrs. San^e of this culture was added to YEPS containing 20% sugar. The flasks w&et iseubated with shaking at 38°C for 48 hours. Samples were withdrawn at dgular intervals and sugar contents and alcohol produced were checked l^ ^andard anthrone and potassium dichromate methods respectively. Example 2 A strain of Saccharomyces cesevimae MTCC YOOOi was grown in YEPD (yeast extract, peptone, dextrost, dktilled water) medium at 30°C for 24 hoiirs and cells were harvested. The sells were treated with lithium acetate and DNA mediated transformation wai caaied 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 designad as T2. This stram T2 was spread over sporalation medium and incubated at 25°C for 3 days. The asci were harvested, treated with zymolyase andspoBes (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 andwere designated as T2-2A and T2-2C. They are haploid and are of a and atpM^ mating type respectively. Strain T2-2C was mutagenised with a chemical mutagen ethyl methane sulfonate and the mutagenised population was subjected to ennchment and selection for fermentation ability at hi\|^ temperature m YEPS medium (Yeast extract. Peptone, Sucrose). Fermentati\|p, 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 enriclpaeat for thermotolerant mutant was repeated a total of six tunes. After final round of fermentation, cells were punfied 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 etheso production at this temperature was selected and designated as MTCC Y0045. Liquid (YEPD) culture of the improved strain MTCC Y0045 was inoculated to molasses medium containing 20% total sugar and incubated with shakmg at 38°C for 16 hours. This ineculum was added to a fresh molasses medium containing 20% total sugar ^d iicubated at 38°C. Incubation was carried out with shaking at 150 rpm fot48 hours. Samples were withdrawn at regular intervals and alcohol produci^ was estimated. Sugar contents and alcohol produced were determined hy standard anthrone and potassium dichromate methods respectively Example 3 A strain of Saccharomyces cermkae T2-2C as described in Example 1 was grown m YEPD for 20 hours at SO'C. The culture was treated with ethidium bromide and from the treatd population of cells, a respiratory deficient mutant, designated as T2-2C4) was selected. This strain is haploid, can not grow on glycerol and is resistat to an antibiotic G418. This strain was crossed with another strain YNN2I7 (haploid, can grow on glycerol and is sensitive to G418; obtained from Prof. Rcnald 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 resistaince and respiratory deficiency. Haploid G418 sensitive cells without respiratoift 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 diptoid cells in the first place. Such back-crossing was earned out a total of six times. This ensured tliat final progeny has most of the genetic material of strain T2-2C and mcorporate desired ho character from the other parent. The strain finally selected is designated as 38-2C. This stram is a stable haploid and ig seasitive to G418. Strain 38-2C was mutagemsed with a chemical mutagen ethyl methane sulfonate and the mutagemsed popullation was subjected to ennchment and selection for fermentation ability at hig\| temperature in YEPS medium (Yeast extract. Peptone, sucrose). Growth auilfciMientation, as determined by loss of weight of the flasks, was monitored as rgular intervals. Once fermentation was complete, 3 ml of this culture was wided to 30ml of fresh YEPS and fermentation was earned out at 37°C as before. This ennchment 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 then- ferme^iation ability and viability at high temperature (38°C). The resulting stnia showing good viability and alcohol production at this temperature was seiei^ed 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 stindard anthrone and potassium dichromate methods respectively. Example 4 A strain of Saccharomyces cermisae T2-2C as debcnbed in Example 1 was grown m 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 T2-2C4) was selected. This strain is haploid, can not grow on glycerol and is resisteit to an antibiotic G418. This strain was crossed with another strain YNN217 (haploid, can grow on glycerol and is sensitive to G418; obtained from Pref. Ronald Davis, Stanford Uruversity, 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 v«th zymolyase, spores (four spores per ascus) v/ere separated with a micromanipulator and individual spores were tested for G418 resistatise aad respiratory deficiency. Haploid G418 sensitive cells without respiratoif 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 m the first place. Such back-crossing was earned 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 sirain finally selected is designated as 38-2C. This strain is a stable haploid and is sensitive to G418. Strain 38-2C was mutagemsed with a chemical mutagen ethyl methane sulfonate and the mutagenised popuft\|tion was subjected to eimchment and selection for fermentation ability at high temperature in YEPS medium (Yeast extract, Peptone, sucrose). Growth and feraentation, as determined by loss of weight of the flasks, was monitored at regjilar intervals. Once fermentation was complete, 3 ml of this culture was added to 30ml of fresh YEPS and fermentation was earned out at 37°C as before. This ennchment 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 fermeniation 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 irafroved strain MTCC Y0046 was inoculated to molasses medium contmtftng 20% total sugar and a part of the culture was incubated with shaking at 38°C for 16 hours, the other at of 30°C for the same penod. Inoculum made at 38°C was added to a fresh molasses medium containing 20% total sugar and indubated at 38°C, the other inoculum grown at 30°C was added to molasses \|20% total sugar) and incubated at 30° C. Incubation was earned 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 alcdiol produced were determined by standard anthrone and potassium dichr^date methods respectively. Example 5 A strain of Saccharomyces cemvimae MTCC YOOOl was grown in YEPD at 30°C for 24 hours. The qpMs were transferred to a sporulation medium and incubated for 3 days. Cells from sporulation medium were treated with zymolyase. Individ^l 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 micromanipolator and placed next to a spore (already arranged m rows) touching it. Mtting between the spores and cells were followed microscopically. Diplords were selected and they were sporulated and spores were separated as descnbed above. The spores were allowed to grow on YEPD and cells which remamed 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 containinig 18% total sugar and incubated with shaking at 38°C for 16 hours. This infeolum was added to a fresh molasses medium containing 18% total sugar a0 intubated at 38°C. Incubation was earned out with shaking at 150 rpm for 8 hours. Progress of fermentation was monitored m 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 cewmdae MTCC Y0046 was grown in YEPD. Another strain MTCC Y0002 #of^osite mating type was also grown in YEPD. These two cells are mix«#. The mixture was then spread on complete Yeast Nitrogen Base GlucosSs»#4ium and incubated at 25°C. Cells from the above plates are harvested aHl then spread on Yeast Nitrogen Base Glycerol medium containing an antibiffliG. Cells growing on this medium are considered to incorporate desired psiperMes from both the parents since neither of the parents can grow on tiis medium. Cells from the selective plates were punfied to single colonies. Pure cultures were then inoculated in 2ml YEPD medium in 24 well microtime 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 mto haploid cells. The spore clones were tested for flocculation as described above. From among these clones strain TF2-9C was selected aai was crossed with MTCC Y0046. A diploid strain ongmating 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 spares were individually tested for the presence of desired properties. The realising strain was found to be flocculent, thermotolerant and is efficient in fermentation at high temperature and designated as MTCC Y0049. Liquid (YEPD) culture of th# improved strain MTCC Y0049 was inoculated to molasses medium contaiqiog 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 penod. Inoculum made at 38°C was added to a fresh molasses medium containing 18.2% total sufp- aad incubated at 38°C, the other inoculum grown at 30°C was added to molasses and incubated at 30°C. Incubation was earned out with shakfeg at 150 rpm for 24 hours. Alcohol production at both temperatures was awanitored. Sugar contents and alcohol produced were determmed by standasi aathrone and potassium dichromate methods respectively. Advantages of the Invention 1. The improved strains of yeast produced are thermotolerant and are capable of fermentation at highM temperature. 2. Viability of the unproved strain! during high temperature fermentation is more than conventional yeast strains. As a result, m case of nse of temperature in the fermenter due to high ambient temperature, power failure, break-down of cooling #vice etc., the process will continue to completion and valuable resourois will not go waste as it happens with conventional yeast. 3. One improved strain of yeast proiuced has additional property of flocculation and as a result, majcmty of the cells sediment at the bottom when agitation is stopped. 4. In addition, the improved yeast strains produced are more osmotolerant, more ethanol toltmnt and as a result, produce higher level of alcohol at normal as weB as at higher temperature compared to conventional yeast strains wjAout any loss of their ability for conversion of sugar to alcohol. 5. Because of above said properties of tiie 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 contant in the wash, which is achieved by high gravity fermentation, there is substantial reduction in steam consumption in the recovery psocess. Net saving of steam m distillation process can be between 0.8 kg to 1.2kg per litre of alcohol distilled. 7. Consequent to high gravity fermementation, there is a net reduction in effluent volume. This may resiit 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. An improved process for the production of alcohol, characterized in fermentation of sugar at high temperature as ddfined herein , using improved thermotolerant flocculent strain of yeast ( Saccherontyces cereviceae) having characteristic such as herein described , the said process comprises fermenting molasses medium containing 20% total sugar by inoculating liquid culture ( in Yeast extract peptone Dextrose mediura) of said thermotolerant strain of yeast in the said molasses medium at a tempeapire 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 alcohol by gravity collection method with yield percentage ranging between 7-11.6% v/v. 2. An improved process for the produoction of alcohol, characterized in fermentation of sugar at high temperature , using improved therrhotolerant flocculent strain of yeast ( Saccharomyces cerevicane) , substantially as herein described with reference to examples .

Full Text

The invention relates to an imroved process for the production of alcohol using an improved thermotolerant flocculent strains of 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 seprepared 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 vanous kinds of renewable fermentable substrates be it (i) sugar (from sugar-cane, sugar beet, fruit) which may be converted to ethanol directly, (11) 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 sagars. (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 geaecrally 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 m the broth without sacnflcing 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 fermenter 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 electncal break down in the plant.
c) saving m energy/steam requirement during distillation; energy being the major vanabk cost in distiliay agemtion.
d) reduction in the volume of effluent.
t) in case of flocculent yeast strains, fermentation may be done by recycling of cells, m a contjasious mode or by conventional batch mode. Because of their flocc«|Mit nature, cells are easily separated firom the fermented broth and m a recycling mode or in a continuous mode less sugar is expected to fee required for building up of biomass.
In conventional method of eth^gpl production, initial concentration of sugar in the broth is kept between 14 to 16 percent. Sugar concentrations higher than this is detrimental to grovM^ of yeast strains used m conventional process and fermentation is thus affected. After completion of fermentation, 6.5 to 8 percent alcohol is obtained in #ie wash. Alcohol is then recovered by distillation using steam.
In order to achieve higher emmol level in the wash, initial sugar concentration in the broth should ate) be higher, thereby increasing the osmolarity of the medium which is detim«atal 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 charactenstic.
One of the drawbacks in convfntional yeast strains is their sensitivity to high temperature. High temperature m 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 devif e, fermentation does not get completed resulting m wastage of material and other resources.
Moreover, conventional yeast strains fail to grow and become inactive at high sugar concentration durmg 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 ethmol. Concurrent with this problem conventional yeasts will not be effective in the fermentation, if ethanol concentration is increased m the both. 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, separatiiip of conventional yeast strains fi-om fermented broth is not easy and may mwt be very efficient in a cell-recycling or continuous mode of operation.
Realising the need to have strains possessing vanous desirable properties and useful for high density fermentation and in a wide range of operatmg temperatures we continued OAir research with the objective of developing thermotolerant strains of yeast which, in addition to tolerating high initial sugar concentration, / e. hjR'iflg osmotolerant charactenstic, could
survive in the higher concentration of ethanol it produces m the broth. In other words, the strain to be used should be tolerant to high concentration of ethanoi produced during the fermentation. The strains thus developed are useful in batch fermentation but can be adopted for contmuous operation as well. For use in a versatile mode of operations, ioceulent characteristic in the strain is also desirable, In order to run fermeniation m cell-recycling or continuous mode, it is desirable that the strain is of floeculent type m addition to retaining other desirable properties. Envisaged advatage of such a unique strain is that less sugar is utilised to build-up the bumass and the sugar thus saved may be converted to ethanoi. Moreover, if a strein is flocculating there is less washout of the cell dunng operation of the process. Such a strain should also give higher productivity of ethanoi. In addtion, high concentration of ethanoi in the wash results in a reduction in steam requirements for distillation. Since the strains used in conventional distillenss make 6.5 to 8% (v/v) alcohol in the wash and have a fermentation efficient of 82.9-86.90 per cent as reported by Arbatti, S.V. and Kale, V.M. in the Pioccedings of International Seminar on "Modernization of Distilleries and Breweries," (organised by All India Distillers Association, July 1989) we concentrated our research for the development of improved strains Of Saccharomyces cerevisiae which will have increased themiotolerance asri floeculent characteristic and will overcome other problems associated with the use of conventional yeast, and make 7 to 12% (v/v) or more alcofcol in the wash with a fermentation efficiency which is as good as, if not beiter than the conventional process.
The main objective of the preSiBit invention is, therefore, to provide a process for the preparation of imprisred thermotolerant strains of genus -
Saccharomyces particularly species cerevisiae which are useful for fermentation process for the preparationof ethanol.
Another objective of the present invention is to introduce the flocculation charactenstics in seme of the said thermotolerant strains which are useful for the preparation of ethanol.
Another objective of the presejaiavention is to produce thermotolerant strams of the genus Saccharomyces spicies - cerevisiae by using the genetic engmeenng, mutation, ennchment and as peated selection processes.
Another objective of the presentinvention is to introduce the flocculent charactenstics m the thermotolerant strains of the genus- Saccharomyces species - cerevisiae by genetic hybridusartion or cytoduction followed by selection.
By thermotolerant it is meani that the strain should be able to efficiently ferment sugars to ethanol at t 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 comman process employed so far to obtain thermotolerant strains of yeast is to swreen for such strains m the natural environment (D'Amore T et a/., 1989, Enzyme Microb Techno! 11: 411-416; Banat IM & Marchant R, 1995, World J. Microbiol Biotechnol 11: 304-306; Banat IM et al, 1992, World J. MicroMol Biotechnol 8: 259-263; Hacking AJ et a/.. 1984, Appl Microbiol. Biotech. If: 361-363; Kida et al, 1992, J. Ferment & Bioeng. lA: 169-173; Laluce C et aL,1991, BiotechnolBioeng 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 method often used to combme thermotolerance with other good proparties by protoplast fusion of strams having such properties (Kida et a/., 1912, J, Ferment & Bioeng. 74: 169-173; Seki T et a/., 1983, Biotechnol Lett 5: 351-356; Stewart GG et a/, 1988, US patent No. 4,772,556). However, the disadvantage here is that unwanted features from the parent strains will alto come into the hybrid strain and there is no method by which those can be readily removed from the hybnd.
Thus the present invention relies to a process for the preparation of thermotolerant strains of yeast which obviates the drawbacks detailed above and thermotolerant mutants can be efetaiaed starting from existing diploid homothailic strains, and the thermoterance 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 homothailic, i.e. haploid cells denved from spores can become diploid by mating with opposite matmg-type (which onginate in the papulation by switching over of mating-type). It is difficult to isolate mutantsirom 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, therefase, essential to generate stable haploid cells for generating mutant strams. Once desired mutants are obtained they

can be further manipulated for construction of other strains with novel charactenstics.
In general, desirable genetic proerties of two strains or more may be brought together, for creating novel steams by many ways such as, mating, (also known as crossmg, genetic hybridisation), cytoduction, protoplast fusion as well as by genetic engmeering and expression of the cloned gene(s). hi doing this manipulations in the laborarory, the strains need to be properly marked and an innovative method has to be developed for the production of improved and novel strams.
Accordingly, the present invention provides a process for the preparation of improved thermotolerant flocculent strains of Saccharomyces, which composes
a) growing a diploid homothallic strain of Saccharomyces, having features such as osmotolerance and ethanol tolerance, in a conventional medium, mutating the homothaftic 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 thensfcetoierant strams retainmg all other properties of the parent strain such as osmotolerance and ethanol tolerance,
c) growing a haploid stram 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 checkmg for ploidy, prototrophy, antibiot resistance and flocculation,
f) sporulatmg the hybnd cells obttfcied in step (e) by known methods and separating individual spores from ascus using conventional methods,
g) mating haploid ceils 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) sporulatmg the cells obtained a step (g) and isolating the spores by known methods, and testing the resultant haploid cells for thermotolerance, flocculation and other desirable properties,
i) mating haploid cells obtained instep (h) with cells obtamed m step (b),
sporulatmg the resultant diploid aad checking the resulting haploid cells for thermotolerance, flocculation and other desirable properties to get improved stable strains of thermotolerant and flocculent Saccharomyces useful for imprved fermentation of fermentable sugars at high temperature
The homothallic strain of Saccharamyces cerevisiae used in step (a), deposited at Microbial Type Culture Collection and designated as MTCC YOOOl, has the following properties: 1 is homothallic, diploid, prototrophic, osmotolerant, non-flocculent, and makes 10 to 12% (v/v) alcohol at 30°C,
This has been deposited at NCYC and tes the accession no. NCYC 2646.
The flocculent strain used in itep (c), deposited at Microbial Type Culture Collection and designated as MTCC Y0002, and also having ATCC accession no. ATTC 90506, has the ftilowing properties: It is heterothallic, haploid, auxotrophic, flocculent, and preduces 2 to 3 % (v/v) alcohol at 30°C.
In a preferred embodiment the growth of MTCC YOOOl may be effected in a known medium such as YIPD (Yeast extract. Peptone, Dextrose) at a temperature m the range of 15°C to 35°C for a penod of 1 to 10 days.
In another preferred embodiin^t the HO gene in MTCC YOOOl may be mutated by conventional mutagenesfe or by targeted gene disruption or by genetic hybndisation method.
In yet another preferred embocttwent the medium used for sporulating the diploid strains such as YOOOl may consist of Agar, Yeast extract. Dextrose, Potassium acetate, and distilled water and the lytic enzyme used may be selected from lyticase, glusulas or zymolyase.
In yet another preferred embodiment the mutagen used m step (b) may be selected from conventional chemicsi 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 embodlEacat fermentation may be earned 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°C to 40°C.
In yet another preferred embodiment the thermotolerant strains obtained in step (b) can be used as such for the fermentative production of 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 strams may be used.
In yet another preferred embodiment the haploid strain in step (c) is grown in a known medium such as YHPD (Yeast extract, Peptone, Dextrose) at a temperature m the range of 15°C to35°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-celll 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 m the range of 1 to 10 days. The medium used in step (e) may be selected from SD Medium (^ast nitrogen base, dextrose, agar and distilled water), YPD (Yeast extract, feptone, Dextrose, Agar and distilled water) or YPG (Yeast extract. Peptone, Oiyeerol, Agar and distilled water).
In yet another preferred embo^ment the medium used in step (e) for isolating diploids may be a convenSpnat medium such as SDG (Yeast nitrogen base, Dextrose, Glycerol, Alp- and distilled water) fortified with broad range antibiotic such as genitici% o%omycin, chloramphenicol and the likes.
In yet another preferred embodiment the different strains may separated by conventional methodb like streaking or dilution plating or by micromanipulation.
In yet another preferred embodimeent 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 stram designated as MTCC YOOOl is homothallic, diploid, prototrophic, osmotolerant, non-flocculent, and makes 10 to 12% v/v alcohol at 30°C. It can sporulate and poduce haploid spores of a and alpha mating types. These spores spontanususly become diploids by switching mating types, and by subsequent hytoidisation of cells of opposite matmg types. To obtain stable haploid derivsatives, the HO gene ( haploid gene ) of this strain, responsible for homothalliasn cr persistent diploidy, was replaced by a mutated version (ho:neo, where it is disrupted by neo gene confemng resistance to antibiotic G418; van Zyl et al., 1993, Current Genetics 23: 290-294) by DNA mediated transformatiin. The transformants were selected on G418 containing medium. One of the resulting strains designated as T2 (Transformant no. 2) was sporulated on apppropriate medium and spores (four per ascus) were separated with the help of a micromanipulator Two of these four spores were sensitive to the an^iotsc 0418 and became diploid. The other two spores which were resistaaat to the said antibiotic and remained haploid were designated as T2-2A an# T2-2C. While T2-2A is of a mating type, T2-2C is of alpha mating t>pe. From these two strains respiratory deficient mutants which can not grow m gij'cerol 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 ai,1992, Proc. Natl. Acad. Sci. 89: 11589-11593; obtained from Prof Ronald Davis, Stanford University, USA) and diploids were sellGted. The selective medium was such that the original two strams (T2-2C-0 aad YNN217) should not survive. Diploids only mcorporatmg 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 earned 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 charactenstics. The haploid strain with desired characteristics was crossed again (back crossing) with T2-2C-0 and sperulated and spores analysed. This back crossing was done a total of six times to ensure retention of all the desired properties and finally two hapled stems 3 8-2A and 38-2C, having the ho gene ( haploid gene) from strain YliN2I7, and of mating types alpha and a, respectively, were selected.
Two strains T2-2C and 38-2C itesciibed above were mutagenised with ethyl methane sulfonate and the mutagenised population was subjected to selection at high temperature fermentSton m YEPS (Yeast extract. Peptone, Sucrose 18% w/v). After fermentatio» was complete, a portion,of the cells were inoculated to fresh YEPS and feainentation was carried out as descnbed above. This process was repeated six times and cells from last round of fermentation was plated out and mdividual colonies were tested for fermentation m YEPS at 38°C. Two stpms designated as (MTCC Y0045) and
(MTCC Y0046) denved from T2-2C and 38-2C, respectively, were finally selected.
These two strains are stable kaploid, thermotolerant, produce more ethanol at 38°C and retain higher vialilty at 38°C compared to the onginal parent strain MTCC YOOOl. Genetic analysis showed that strain MTCC Y0045 is of alpha mating type and stain 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 haloid thermotolerant strains were also constructed by genetic crosses. Strati MTCC Y0046 (a mating type) was crossed with a strain 38-2A (alpha nKiing type and non thermotolerant). The resulting diploid strams were sporuliiled and spore clones were analysed as stated earlier. A thermotolerant cloae of alpha mating type (106-2A) was selected and mated with MTCC Y0046. The resulting diploid strain designated as MTCC Y0047 was foui^ to have high temperature fermentation ability comparable to haploid MTCC Y0946. In another strategy, spores of MTCC YOOOl were separated individually with the help of a micromanipulator and cells of MTCC ¥0046 were placed next to these spores for mating, which was monitored undfr 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 populatiea. Few such diploids were analysed. One strain designated as MTCC Y0048 was found to have thermotolerance
and high temperature fermentation elfeiency comparable to onginal MTCC Y0046 haploid strain.
In order to construct further improved strains having characteristics such as osmotolerance, ethanol tolerace, flocculation and capable of fermentation at high temperature, haploid strain MTCC Y0046 was crossed with another haploid strain designated as MTCC Y0002 as descnbed earlier. Cells of these two haploid strams were placed next to each other m pairs, incubated till colonies appeared on plaHs and the colonies were then streaked on non-selective medium. Single colonies were picked and checked for desirable properties on appropnate sel#tive medium. The selective medium was designed such that the original tma strains (MTCC Y0046 and MTCC Y0002) would not survive. Only hylids created by transfer of desirable genetic matenal from both MTCC Y0006 aad 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 stfaint designated as TF5 having desired properties was chosen. The strain TFS was sporulated and the spores were analysed and one spore clone named TF5-5B having desired characteristics was crossed with MTCC Y0046 and m d^oid strain designated as TF6 was obtained.
TF6 was again sporulated and 8#er separation of individual spores and their characterisation, a strain MTCC Y{X)49 was selected. This strain is haploid, thermotolerant, flocculent mi is efficient m fermentation at high temperature.
The improved strams have b^i deposited m the National Facility on Microbial Type Culture Collection and Qene Bank (MTCC) located at the Institute of Microbial Technology, a constituent laboratory of Council of Scientific and Industnal Research, Tliey have been assigned the following accession numbers:
MTCC Y 0045 haploid, thermotflerant, mutant denvative of T2-2C
MTCC Y0046 haploid, thennol»lerant, mutant denvative of 3 8-2C
MTCC Y0047 diploid, thermoteierMit, derived fi:om 106-2AxY0046
MTCC Y0048 diploid,, thermoi»lerant, derived fi-om Y0046xY0001
MTCC Y0049 haploid, thermoirferant, flocculent derived fi-om TF6
The improved strains listed abmc have the following charactenstics:
(i) they grow at a temperature ranging between 15°C and 38°C m Yeast Extract Peptone Dextrose (YEPP) medium with 2% glucose,
(ii) they grow on agar plates contaKling 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) ttiey also grow m Yeast Extract, Peptone, Dextrose (YEPD) medium in presence of up to 12% e^aaol. The strains are, therefore,
osmotolerant, ethanol tolerant, fewiotolerant and produce high level of alcohol at temperature betwwsi 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.
vu) they retain their viability and feiRientation ability for use m recycling.
viu) under appropnate conditions of flocculation MTCC Y0049 settle or sediment within 10 seconds to S5 seconds.
ix) properties descnbed 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, sacciiarose and raffinose as carbon sources but do not grow in salicm, lactose, inositol, citrate, 2-Keto-D-glucorate, arabinose, xylose, adanitol, xylitol, KirbMol, methyl-D-glycoside, n-acetyl-glucosamme, cellobiose, trehalose an4 melizitose. Growth was poor when sodium nitrate, potassium nitrate or lysme were used as a sole source of nitrogen.
Molasses is a by-product of sugar manufactunng process. What remains after sugar is extracted from aigar 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 Gade C molasses on the other hand, has
about 40% (w/v) sugar. In order to achieve a desired sugar concentration in a fermenter, molasses is accordingly diluted with water.
Since ethanolic feraientatioft is a process by which certain microorganisms convert sugars such ai sucrose, glucose and fructose to ethyl alcohol; the sugars used in the process of the present invention can be glucose, sucrose, fructose or other reducing suprs as such or as present m molasses or a combination of these or the sugars obtained from starch and other lignocellulosic matenal.
Since conventional yeast straiae prefer a temperature of around 30°C for fermentation, the fermenters are ptmndnd with a cooling device to maintain the temperature. Rise in temperature due to breakdown of cooling device, power failure, heat generated due to mstabolic activities of yeast or due to any other cause results in inefficient or ccjfnplete stoppage of fermentation. As a result, valuable resources are waste!. This type of problem can occur throughout the year specially m summer when ambient temperature may go well over 40°C m many parts of the country. The strains developed in the present invention are capable of fermeatation at normal temperature as well as higher temperature. Thus, fermentati«i 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 dunng fermeaction. The strains also ferment sugars at temperatures lower than 38°C with equal efficiency.
Accordingly the present invention povides an improved process for the production of alcohol using improved sfrain of Saccharomyces cereviceae which composes growing thermotoloment flocculent strain of Saccharomyces having charactenstic such as herein described , in liquid YEPD Yeastextractpeptone Dextrose) medium, moculating the liquid culture of said Saccharomyces strain to molasses medium containing 20% total sugar , cultunng at a temperature ranging 15-40° C for 16 -96 hrs , addmg moculum of obtained culture m fresh molasses a|edi»m at 38 ° C for 48 hrs at 150 rpm ,recovenng the alcohol produced with yeild percentage ranging between 7-11.6%v/v.
It could be observed from the descnpfei 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 ; 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 YOOOl was grown in YEPD (yeast extract, peptone, dextrose, distilled water) medium at 30°C for 24 hours and celbwfflse harvested. The cells were treated with lithium acetate and DNA mediafted transformation was carried out as descnbed by Kaiser, et al (1994, Method in Yeast Genetics, CSHL, Press, USA). Transformants were selected on YEPD medium containing an antibiotic G418. One transformant was sdected and designated as T2. This stram 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 ofa. nacromanipulator. Individual spores were checked for G418 resistance, phidy and mating types. Cells denved
from two spores having antibiotic instance 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 mutagenisedwith a chemical mutagen ethyl methane sulfonate and the mutagenised population was subjected to ennchment and selection for fermentation ability at high temperature m YEPS medium (Yeast extract, Peptone, Sucrose). Fermentialsn, as determmed 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 fr«ib YEPS and fermentation was earned out at 37°C as before. This enri4tt«ait for thermotoierant mutant was repeated a total of six times. After fteai round of fermentation, cells were punfied to single colonies on YEPD and were individually tested for their fermentation ability and viability at Ifeh temperature (38°C). The resulting strain showing good viability and etNinol production at this temperature was selected and designated as MTCC Y0W5.
The improved strain was inoci4iat©d in YEPD medium and incubated at 30°C with shaking for 24 hrs. San^e of this culture was added to YEPS containing 20% sugar. The flasks w&et iseubated with shaking at 38°C for 48 hours. Samples were withdrawn at dgular intervals and sugar contents and alcohol produced were checked l^ ^andard anthrone and potassium dichromate methods respectively.
Example 2
A strain of Saccharomyces cesevimae MTCC YOOOi was grown in YEPD (yeast extract, peptone, dextrost, dktilled water) medium at 30°C for 24 hoiirs and cells were harvested. The sells were treated with lithium acetate and DNA mediated transformation wai caaied 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 designad as T2. This stram T2 was spread over sporalation medium and incubated at 25°C for 3 days. The asci were harvested, treated with zymolyase andspoBes (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 andwere designated as T2-2A and T2-2C. They are haploid and are of a and atpM^ mating type respectively.
Strain T2-2C was mutagenised with a chemical mutagen ethyl methane sulfonate and the mutagenised population was subjected to ennchment and selection for fermentation ability at hi|^ temperature m YEPS medium (Yeast extract. Peptone, Sucrose). Fermentati|p, 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 enriclpaeat for thermotolerant mutant was repeated a total of six tunes. After final round of fermentation, cells were punfied 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 etheso production at this temperature was selected and designated as MTCC Y0045.
Liquid (YEPD) culture of the improved strain MTCC Y0045 was inoculated to molasses medium containing 20% total sugar and incubated with shakmg at 38°C for 16 hours. This ineculum was added to a fresh molasses medium containing 20% total sugar ^d iicubated at 38°C. Incubation was carried out with shaking at 150 rpm fot48 hours. Samples were withdrawn at regular intervals and alcohol produci^ was estimated. Sugar contents and alcohol produced were determined hy standard anthrone and potassium dichromate methods respectively
Example 3
A strain of Saccharomyces cermkae T2-2C as described in Example 1 was grown m YEPD for 20 hours at SO'C. The culture was treated with ethidium bromide and from the treatd population of cells, a respiratory deficient mutant, designated as T2-2C4) was selected. This strain is haploid, can not grow on glycerol and is resistat to an antibiotic G418. This strain was crossed with another strain YNN2I7 (haploid, can grow on glycerol and is sensitive to G418; obtained from Prof. Rcnald 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 resistaince and respiratory deficiency. Haploid G418 sensitive cells without respiratoift 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 diptoid cells in the first place. Such back-crossing was earned out a total of six times. This ensured tliat final progeny has most of the genetic material of strain T2-2C and mcorporate desired ho character from the other parent. The strain finally selected is designated as 38-2C. This stram is a stable haploid and ig seasitive to G418.
Strain 38-2C was mutagemsed with a chemical mutagen ethyl methane sulfonate and the mutagemsed popullation was subjected to ennchment and selection for fermentation ability at hig| temperature in YEPS medium (Yeast extract. Peptone, sucrose). Growth auilfciMientation, as determined by loss of weight of the flasks, was monitored as rgular intervals. Once fermentation was complete, 3 ml of this culture was wided to 30ml of fresh YEPS and fermentation was earned out at 37°C as before. This ennchment 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 then- ferme^iation ability and viability at high temperature (38°C). The resulting stnia showing good viability and alcohol production at this temperature was seiei^ed 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 stindard anthrone and potassium dichromate methods respectively.
Example 4
A strain of Saccharomyces cermisae T2-2C as debcnbed in Example 1 was grown m 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 T2-2C4) was selected. This strain is haploid, can not grow on glycerol and is resisteit to an antibiotic G418. This strain was crossed with another strain YNN217 (haploid, can grow on glycerol and is sensitive to G418; obtained from Pref. Ronald Davis, Stanford Uruversity, 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 v«th zymolyase, spores (four spores per ascus) v/ere separated with a micromanipulator and individual spores were tested for G418 resistatise aad respiratory deficiency. Haploid G418 sensitive cells without respiratoif 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 m the first place. Such back-crossing was earned 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 sirain finally selected is designated as 38-2C. This strain is a stable haploid and is sensitive to G418.
Strain 38-2C was mutagemsed with a chemical mutagen ethyl methane sulfonate and the mutagenised popuft|tion was subjected to eimchment and selection for fermentation ability at high temperature in YEPS medium (Yeast
extract, Peptone, sucrose). Growth and feraentation, as determined by loss of weight of the flasks, was monitored at regjilar intervals. Once fermentation was complete, 3 ml of this culture was added to 30ml of fresh YEPS and fermentation was earned out at 37°C as before. This ennchment 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 fermeniation 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 irafroved strain MTCC Y0046 was inoculated to molasses medium contmtftng 20% total sugar and a part of the culture was incubated with shaking at 38°C for 16 hours, the other at of 30°C for the same penod. Inoculum made at 38°C was added to a fresh molasses medium containing 20% total sugar and indubated at 38°C, the other inoculum grown at 30°C was added to molasses |20% total sugar) and incubated at 30° C. Incubation was earned 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 alcdiol produced were determined by standard anthrone and potassium dichr^date methods respectively.
Example 5
A strain of Saccharomyces cemvimae MTCC YOOOl was grown in YEPD at 30°C for 24 hours. The qpMs were transferred to a sporulation medium and incubated for 3 days. Cells from sporulation medium were treated with zymolyase. Individ^l 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 micromanipolator and placed next to a spore (already arranged m rows) touching it. Mtting between the spores and cells were followed microscopically. Diplords were selected and they were sporulated and spores were separated as descnbed above. The spores were allowed to grow on YEPD and cells which remamed 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 containinig 18% total sugar and incubated with shaking at 38°C for 16 hours. This infeolum was added to a fresh molasses medium containing 18% total sugar a0 intubated at 38°C. Incubation was earned out with shaking at 150 rpm for 8 hours. Progress of fermentation was monitored m 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 cewmdae MTCC Y0046 was grown in YEPD. Another strain MTCC Y0002 #of^osite mating type was also grown in YEPD. These two cells are mix«#. The mixture was then spread on complete Yeast Nitrogen Base GlucosSs»#4ium and incubated at 25°C. Cells from the above plates are harvested aHl then spread on Yeast Nitrogen Base Glycerol medium containing an antibiffliG. Cells growing on this medium are considered to incorporate desired psiperMes from both the parents since neither of the parents can grow on tiis medium. Cells from the selective
plates were punfied to single colonies. Pure cultures were then inoculated in 2ml YEPD medium in 24 well microtime 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 mto haploid cells. The spore clones were tested for flocculation as described above. From among these clones strain TF2-9C was selected aai was crossed with MTCC Y0046. A diploid strain ongmating 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 spares were individually tested for the presence of desired properties. The realising strain was found to be flocculent, thermotolerant and is efficient in fermentation at high temperature and designated as MTCC Y0049.
Liquid (YEPD) culture of th# improved strain MTCC Y0049 was inoculated to molasses medium contaiqiog 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 penod. Inoculum made at 38°C was added to a fresh molasses medium containing 18.2% total sufp- aad incubated at 38°C, the other inoculum grown at 30°C was added to molasses and incubated at 30°C.
Incubation was earned out with shakfeg at 150 rpm for 24 hours. Alcohol production at both temperatures was awanitored. Sugar contents and alcohol produced were determmed by standasi aathrone and potassium dichromate methods respectively.
Advantages of the Invention
1. The improved strains of yeast produced are thermotolerant and are capable of fermentation at highM temperature.
2. Viability of the unproved strain! during high temperature fermentation is more than conventional yeast strains. As a result, m case of nse of temperature in the fermenter due to high ambient temperature, power failure, break-down of cooling #vice etc., the process will continue to completion and valuable resourois will not go waste as it happens with conventional yeast.
3. One improved strain of yeast proiuced has additional property of flocculation and as a result, majcmty of the cells sediment at the bottom when agitation is stopped.
4. In addition, the improved yeast strains produced are more osmotolerant, more ethanol toltmnt and as a result, produce higher level of alcohol at normal as weB as at higher temperature compared to conventional yeast strains wjAout any loss of their ability for conversion of sugar to alcohol.
5. Because of above said properties of tiie 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 contant in the wash, which is achieved by high gravity fermentation, there is substantial reduction in steam consumption in the recovery psocess. Net saving of steam m distillation process can be between 0.8 kg to 1.2kg per litre of alcohol distilled.
7. Consequent to high gravity fermementation, there is a net reduction in effluent volume. This may resiit 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. An improved process for the production of alcohol, characterized in fermentation of sugar at high temperature as ddfined herein , using improved thermotolerant flocculent strain of yeast ( Saccherontyces cereviceae) having characteristic such as herein described , the said process comprises fermenting molasses medium containing 20% total sugar by inoculating liquid culture ( in Yeast extract peptone Dextrose mediura) of said thermotolerant strain of yeast in the said molasses medium at a tempeapire 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 alcohol by gravity collection method with yield percentage ranging between 7-11.6% v/v.
2. An improved process for the produoction of alcohol, characterized in fermentation of sugar at high temperature , using improved therrhotolerant flocculent strain of yeast ( Saccharomyces cerevicane) , substantially as herein described with reference to examples .

Documents:

1112-del-1998-abstract.pdf

1112-del-1998-claims.pdf

1112-del-1998-complete specification (granted).pdf

1112-del-1998-correspondence-others.pdf

1112-del-1998-correspondence-po.pdf

1112-del-1998-description (complete).pdf

1112-del-1998-form-1.pdf

1112-del-1998-form-2.pdf

1112-del-1998-form-3.pdf

1112-del-1998-form-9.pdf

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Patent Number

189737

Indian Patent Application Number

1112/DEL/1998

PG Journal Number

16/2003

Publication Date

19-Apr-2003

Grant Date

27-Jan-2004

Date of Filing

27-Apr-1998

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	GANDHAM SATYANARAYANA PRASAD	INSTITUTE OF MICROBIAL TECHNOLOGY, CHANDIGARH,INDIA.
2	INDRANI GHOSH	INSTITUTE OF MICROBIAL TECHNOLOGY, CHANDIGARH,INDIA.
3	ROHINI CHOPRA	INSTITUTE OF MICROBIAL TECHNOLOGY, CHANDIGARH,INDIA.
4	TAPAN CHAKRABARTI	INSTITUTE OF MICROBIAL TECHNOLOGY, CHANDIGARH,INDIA.
5	KALIANNAN GANESAN	INSTITUTE OF MICROBIAL TECHNOLOGY, CHANDIGARH,INDIA.
6	VISHVA MITRA SHARMA	INSTITUTE OF MICROBIAL TECHNOLOGY, CHANDIGARH,INDIA.

PCT International Classification Number

C12N 1/12

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA