TEMPERATURE TOLERANCE AND OTHER PROPERTIES OF TWO ETHANOL PRODUCING SACCHAROMYCES CEREVISIAE STRAINS ISOLATED FROM COCONUT TODDY

: Several yeast strains capable of fermenting glucose at 40°(5 were isolated fromcoconut toddy. Two strains, whichgavehighethanol concentration at 40° C were identified as strains of Saccharomyces cereuisiae. Strain T-31 (NCYC 2401) at 40°C gave high alcohol concentration in both YPS and molasses media, 7.4% and 5.4% v/v respectively, while strain SK-33 (NCYC 2402) gave higher concentration of alcohol in synthetic medium, 8.4% vlv. The maximum growth temperature ofboth these strains was45°Cindicatingtheirthermotolerant behaviour. Of these two strains. T-31 was found to be suitable for alcohol fermentation at high temperature (40°C) using molasses, based on parameters such as maximum CO, productivity (0.216 gh-l), final ethanol concentration (7.4% vlv), yield efficiency (0.579) and minimal cell growth rate (0.029 cells ml-I 11-l).


INTRODUCTION
The limited availability of world's crude oil and its constant price increase aroused renewed interest in the gasohol concept. Fuel ethanol has been produced in Brazil since 1933, while both Germany and Japan used it as a source of fuel during the Second World War. Production of fuel ethanol using industrial byproducts such as molasses would be a promising endeavour for developing countries, which do not have any fossil fuels.
Molasses, which is a by-product of cane sugar industry, is mainly utilised in the production of ethanol in the distilleries of Sri Lanka. With the setting up of new sugar factories in the country, there is a considerable increase in the availability of sugarcane molasses. Nevertheless, these distilleries operate at low efficiencies (50 -60%) compared to other distilleries in the wor1d.l This is due to inefficiency in the method of distillation and unsuitability of the yeast strains for fermenting molasses under tropical environmental conditions. The compound effect of high ambient temperature in tropical countries and exothermic fermentation reaction elevate the temperature of the fermeptation mash. High temperature usually inhibits the fermentation ability of the yeast strain used, as most of the conventional yeasts are temperature sensitive. To minimise this inhibition, a cooling system is used to maintain the temperature around 35OC or below. However this process increases the capital and running costs. In view of this situation, tropical fermentation technology as regards ethanol fermentation by yeast, requires strains capable of fermenting sugars efficiently at high temperature of around 40°C. There are certain advantages in high temperature fermentation by yeast. They are, faster rates of substrate consuinption and ethanol formation, facilitation of easy ethanol recovery and savings on cooling cost. As the fermentation is completed within 36 -48 h, loss of ethanol due to evaporation is considerably less even a t temperatures around 40°C. Therefore, use of high temperature tolerant yeast, in fermentation industry under tropical environmental conditions is cost effective. There are several reports on the isolation of thermotolerant strains belonging to different genera. The isolatioi~ of five thermotolerant strains of Klz~yueromyces strains capable of growth and high-level ethanol production, on glucose and molasses fermentation, at temperatures in the range of 45 -50°C had been reported. Klz~yverolnyces nzarxianzrs IMB 3 had produced 35 g/l of alcohol a t 13 h following growth a t 45°C on sucrose containing medium.%ndersonet al. reported an efficient carbohydrate fermenting Klz~yueromyces mal-xianz~s var. nzarxialzus producing 6% w/v ethanol a t 43°C in 24 h.ai Two Saccharolnyces and one Calzdida strains reported by Haclcing et al. were found to meet minimum commercial targets set a t 8% (v/v) ethanol from 14% w/v glucose at 40°C.4 This paper describes some features of two thermotolerant ethanol producing Sacclzaromyces cerevisiae strains isolated from cocollut sap.

METHODS AND MATERIALS
Isolation of high-te~nperatz~re yeast: Enrichment cultures were prepared using 5 m.1 samples of toddy in 45 ml of lo(% glucose and incubated a t 40°C for 24 h. Subsequently the enriched cultures were used,to prepare a series of dilution. followed by plating 0.1 ml of each dilution on Yeast extract -Peptone -Dextrose agar (YPD) medium composed of 2% dextrose, 2% peptone, 1% yeast extract, 2% agar and complete synthetic medium which has a composition similar to the basal medium described."~ both these media 50 ppm of Streptomycii~ was added to inhibit bacterial growth. The plates were incubated a t 40°C for 2-3 days. Morphologically different colonies that appeared on plates were picked up and purified by streaking on fresh YPD plates.
Selection of'yeust strains capable offel-mentingat 40°C: Fermentation tests were carried out with each of the strains isolated using broth cultures. First a seed culture was prepared using YPD broth a t 30°C and the cell concentration of each of these cultures was monitored using a haemocytometer. For the fermentation test, 50ml amounts ofYPD medium containing 15% (w/v) glucose were dispensed in 250-ml capacity Erlenmeyer flasks fitted with fermentation bungs. The fermentation bungs were filled with concentrated sulphuric acid, which permitted only carbon dioxide to evolve from the culture. Water vapour and vaporized ethanol were trapped in the sulphuric acid. The flasks were inoculated with a known amount of cells (1x108 cells ml-I), from the seed culture of each of the isolates and were weighed after inoculation and thenincubated statically a t 40°C for 72 h. The fermentation was monitored by measuring the daily carbon dioxide output as reflected by decrease in the weight of the whole culture. Twice the weight of carbon dioxide evolved is equivalent to the amount of sugar consumed by each yeast strain used in fermentatiom6 Amount of sugar used up by each of the strains in fermentation was calculated and those strains that used up 50% or more of the initial sugar content were selected for further studies.
Fermentation tests were again done using Yeast extract -Peptone -Sucrose (YPS) medium and synthetic medium containing 20% (wlv) sucrose, a t 40°C, to check the fermentation ability of the selected yeast strains with sucrose as the carbon source. For comparison, baker's yeast was also used in these experiments. The amount of carbon dioxide evolved (g) and the final ethanol concentration (% V/V) of the fermenting mash were measured. The final ethanol concentration was measured using an ebulliometer.

Metlzods of identification:
For taxonomical identification of yeast, the methods described5.j were followed.
GI-owtlz at different temperatzrres: The selected strains were tested for their ability to grow a t different temperatures such as 40,45,47 and 50°C inYPD agar medium. In complete synthetic liquid medium, the growth rates of the selected strains at 40°C were compared with Baker's yeast by measuring the optical density at 660 nm, a t intervals and then converted to cell density using a calibration curve.
Fermentation characteristics of selected yeast a t different temnperatrrres: Fermentation tests were carried out with the selected strains and Baker's yeast a t different temperatures, 25,30 and 40°C. As 40°C was found to be the optimum growth temperature of the yeast, it was selected to be the-maximum temperature limit for fermentation. In both YPS and complete synthetic medium 20% (wlv) sucrose was used. The carbon dioxide evolution rate and final ethanol concentration were determined as described earlier.
Fermentation using lnolasses medium : Molasses was diluted three folds to give approximately 20% fermentable sugars and potassium phosphate (0.05%) and magnesium sulphate (0.5%)S were added. Before autoclakg, the pH was adjusted to 4.8. Fermentation was carried out a t 30 and 40°C. The amount of carbon dioxide evolved and the ethanol concentrations (% vlv) were determined as described earlier.
Selecting 212c best yeast st?-ail^ for ilzdz~strial alcoholproductzon: For this purpose, the parameters, maximum productivity (based on carbon dioxide evolution), final alcohol concentration, ethanol yield ratio and maximum growth rate were uscd. Ethanol yield ratio was computed by dividing the actual ethanol yield by theoretical e than01 yield on the basis ofGay-Lussac equation (i.e. 0.511 g ethanol g-' glucose, fructose or sucrose). The maximum growth rate was calcuIated from the exponential phase of the growth curve. Optical density was measured using a spectrophotometer a t 660 a m and then converted to cell density using a calibration curve. Higher values for final ethanol concentration, carbon dioxide evolution rate and ethanol yicld ratio have a positive influence on alcohol productivity while higlier values for growth rate have a negative influence on alcohol prod~ctivity.~

RESULTS
Colonies, which had different morphology on dilution plates, were picked up and purified by streaking on fresh YPD plates. FiReen,stock cultures were prepared using YPD medium. Code numbers identified these.
In the fermentation experiments the loss in weight of thc whole culture rxflected the carbon dioxide productivity and twice that value indicated the amount of' sugar consumed by the cell to produce alcohol according to Gay Lussac equation. Six strains capable of fermenting more than 50% ofinitial sucrose a t 40°C were selected from the stock culture by preliminary fermentation tests. Out of the six strains, only two strains (SK-33 and T-31) gave high alcol~ol yield a t 40°C wit11 sucrose as the carbon source (Table 1). The two strains, T-31 and SK-33 thus selected agreed well with the standard description for Sacckaromyces cerevisiae (Tables 2 and 3). Full DNA finger printing analysis of the above two cultures showed that they are significantly different from other brewing strains held in the National Collection of Yeast Cultures (NCYC) a t Norwich, UK. Therefore, the above cultures, T-31& SK-33 were accessioned into NCYC after assigning the numbers NCYC 2401 and 2402 respectively.    The maximum growth temperat~~re of these two yeasts was 4Ei°C. Both grew very well a t 40°C ( Table 4). No growth was observed at 47OC and 50°C. The growth rate of SK-33 in synthetic medium was higher while T-31 and Baker's yeast had a lower growth rate. The yeasts appear thermotolerant. Alcolzol ferlnentation at different temperatz~res: In fermentation tests a t 30°C in YPS medium, SK-33 and T-31 gave more than 12 % vlv alcohol, whereas Baker's yeast gave less alcohol 11.7% vlv. At 40°C in the same medium, strain T-31 performed better, giving an alcohol concentration around 7.4% vlv (Fig. 1). With increase oftemperature from 30 to 40°C, the alcohol concentration obtained with this strain decreased by about 41%. In the synthetic medium wit11 20% sucrose a t 30°C all strains gave higher alcohol concentration, more than 10% vlv and SK-33 gave highest concentration, 13.2% vlv. At 40°C also, the concentratiolls were higher in this medium with strain SK-33 giving the highest value (8.4% vlv, Fig. 2). In the synthetic medium with increase of temperature the % decrease in alcohol concentration was about 36%. The 'balance sheet' (Table 5) with respect to fate of sugar and production of ethanol in the two media a t 40°C shows that the difference between the observed and calculated values for ethanol is very small. This confirmed that there is no great loss of ethanol due to evaporation a t high temperature. Alcolzol fermentation using ~nolusses: In the molasses medium a t 30 and at 40 OC, T-31 gave highest alcohol concentration of 11.9% vlv and 5.4% vlv respectively (Fig. 3). Baker's yeast in the same medium produced lower alcohol con cent^-ations, 11.5%, 4.7% at 30 and 40°C respectively. The percentage decrease in the alcohol yield with increase oftemperature, was less in the case of T-31(54%; tllan in Baker's yeast (59%). The values obtained for different parameters that were used in the selection of a suitable strain for industrial alcohol production are shown i n Table 6. In strain T-31, all parameters that have a positive influence on alcohol production (i.e. CO, productivity, final ethanol concentration and ethanol yield ratio) gave higher values. Further, the same strain gave a lower value for maximum growth rate (0.029), which has a negative influence on alcohol production.

DISCUSSION
Altl~ough several reports are available on the flora of fermenting coconut palm wine, no studies have been done on thermotolerant yeast strains. Temperature is a paramount important regulatory factor in alcohol fermentation, particularly in tropical countries. There are a number of studies on the thermotolerance of growth and fermentation of different yeast strains. However this is the first instance that apparently thermotolerant yeasts have been isolated from coconut toddy. The strain CT-31) Saccharomyces cerevisiae (NCYC 2401), isolated from coconut toddy gave 7.4% vlv alcohol within 24 to 36 h, in YPS medium, but the initial sugar concentration was higher 20% wlv. Two Saccharornyces and, one Candida have been found to meet minimum commercial targets set a t 8% (vlv) ethanol from 14% wlv glucose at 40°C.4 There have been several studies on the thermotolerance of growth and fermentation of S. cerevisiae and S. i~v a r z~l n strains and some results are contradictory. In general, the conclusion is that higher the fermentation temperature greater the inhibitory effect of ethanol on ~e l l s .~~J~ Some have reported increase in ethanol tolerance with increase of temperature.12 It has also been reported that growth and fermentation can be lost in the range of 35-450C. 13 In this study, at 40°C Saccharomyces cerevisiae (NCYC 2401) gave 7.4% and 5.4 % vlv alcohol in YPS and. molasses media respectively. When alcohol yields a t high temperatures are comparedwith those at lower temperatures, both these yeasts were found to give higher alcohol yields at low temperatures in all media tested. This decrease in alcohol yield at higher temperature could be due to the damaging effect of temperature on yeast cells. However, the results indicate that the yeast strains isolated from coconut toddy have the potential to produce high alcohol yields at fairly high temperatures and would be suitable to be used in molasses fermentation even without a sophisticated cooling system.