Indian Journal of Experimental Biology Vol. 40, March 2002, pp. 325-328

Fermentation of starch to ethanol by an amylolytic yeast Saccharomyces diastaticus SM - 1 0

S Sharma, M Pandey & B Saharan

Department of Microbiology, CCS Haryana Agricultural University, Hisar 1 25 004, India

Received 4 December 2000; revised 8 August 2001

A total of fifteen yeast strains were isolated from natural sources including fruits, soil, molasses, honey and a variety of indigeneous fermented foods. Screening of these strains for growth, ethanol production and glucoamylase activity led to se­lection of a yeast strain SM-l O identified as S. diastaticus having maximum glucoamylase activity (80 units mr') and etha­nol production from starch (3.5%). Ethanol production from wheat flour was found to be 1 .75% which could be increased to 5.2% after treatment of wheat flour with pepsin, diastase and glucoamylase.

There is much interest in the utilization of starch as an alternate source of energy because of its renewable nature and availability throughout the world in large quantities. There are a variety of products that can be obtained from starch biomass via hydrolysis, and ethanol is one of the largest volume of product that can be produced from biomass. Ethanol production from starch involves two steps: (a) hydrolysis of starch to glucose either by amylolytic enzymes or in­organic acids and (b) fermentation of glucose to etha­nol by yeast. In this process the conversion of starch to glucose is an expensive and time consuming step. A system of simultaneous saccharification and fer­mentation has been attempted by use of mixed culture of two yeasts' or mold and yeast 2.3. Certain amy­lolytic enzymes along with yeast have also been used for the production of ethanol from starch in a single step fermentation4•s. The biggest drawback of using this system is that yeast, fungi and enzymes do not have similar optimum cultural or reaction conditions.

Common ethanol producing yeast such as Sac­charomyces cerevisiae, S. carisbergensis, S. sake or the bacterium Zymomonas mobilis, can not utilize starch because they lack the starch splitting enzymes. In the last few years, several amylolytic yeast species belonging to Candida, Lipomyces, Saccharomycopsis, Schwanniomyces, Filobasidium and Trichosporon have been isolated and studied extensively for direct conversion of starchy substrates to ethanol. This is of considerable importance as direct starch fermentation does not require liquefaction and saccharification. Considering these aspects, yeast strains were isolated from natural sources having fermentative as well as ·

amylolytic activity for production of ethanol from starch (Wheat flour) .

Materials and Methods Yeast cultures-In the present investigation 1 5

amylolytic yeast cultures (SM-l to SM- 1 5 ) isolated from natural sources were included.

Media, enzymes and fermentation discs-Cultures were grown at 30°C on starch, yeast extract and pep­tone (SYP) medium slants containing 2% peptone, 2% soluble starch (Merck), 1 % yeast extract, 2% agar6• Pepsin (Sisco Laboratories), bacteriological differentiation discs and diastase (Hi-media) and glucoamylase (Kedia castle Industries, Durg) were used as and when required in the experiments.

Isolation of yeast strains from various sources­Samples of fruits, molasses, stored honey, soil and fermented foods were inoculated in SYP broth for enrichment at 30°C for 24 hr. The enriched samples were streaked on SYP medium plates and incubated at 30°C for 48 hr. The yeast like colonies appeared and these were purified by further streaking on SYP me­dium plates and finally transferred on SYP slopes in tubes� A total of 3 1 samples were plated initially out of which 1 5 samples showed yeast like colonies.

Ethanol production by yeast strains- Ethanol production by isolated strains was determined by stan­dard procedures7• Equal biomass (0.5g wet weight per 1 00 ml) was inoculated in sterilized dextrose fer­mentation medium (DFM) containing 15% dextrose, 0.5% yeast extract, 0.5% peptone (PH 5.0). Fermenta­tion was carried out at 30°C and ethanol produced was estimated. To study ethanol production from starch,

326 INDIAN J EXP BIOL, MARCH 2002

cultures were grown in starch, yeast extract and pep­tone medium (SYP) containing 2% starch and incu­bated on rotary shaker at 30°C. After 24 hr equal number of cells @ 2 x 106 were inoculated into 100 ml starch fermentation medium (SFM) containing starch 10%, peptone 0.5% and yeast extract 0.5% (PH 5 .0). Fermentation was carried out at 30°C and sam­ples were analysed for ethanol content. Ethanol was estimated spectrophotometricalll after distillation of the samples (Fig. 1 ) .

Estimation of glucoamylase activity-The gluco­amylase activity was estimated by the method of Lemmel et aP. Cell suspension with an optical density 0.5-0.7 at 6 10 nm was i noculated into SYP medium containing 2% starch and incubated at 30°C on a ro­tary shaker. Cell free culture supernatant obtained by centrifugation was used for measuring extra-cellular amylolytic activity by incubating 0.5 ml culture su­pernatant with 0.5 ml of 1 % soluble starch (merck) dissolved in 0. 1 M acetate buffer (PH 4.8) at 30°C in a water bath for 3 min to convert starch to sugar which was estimated by DNSA reagent. One unit of amy-

25 per cent wheat flour starch !

Gelatinization by boiling for 5 min on magnetic stirrer

! Saccharification with enzyme for 4 hr at 40°C

! Inoculation of wheat hydrosylate with 1 8 hr old culture

at the level of 1 5 , 30 and 45 per cent !

Fermentation at 30°C in BOD incubator under stationary conditions

! Estimation of ethanol

Fig. 1 -Flow diagram of ethanol production from wheat flour

lolytic activity is defined as the amount of enzyme which release 1 J.!M (0. 1 8 mg) of glucose from starch in 3 min.

Ethanol production from wheat flour-The etha­nol production from wheat flour was carried out after gelatinization and pretreatment of wheat starch 1 0. Ge­latinization of wheat starch was done by open cooking method I I . Wheat flour slurry (25%) was prepared and pH was adjusted to 5 .0. It was cooked by boiling for 5 min on magnetic stirrer with continuous agitation and cooled down to 40°C. Pretreatment of wheat starch was carried out with 1 3 .5 mg (40 units) pepsin and 1 .3 mg ( 1 690 units) diastase per 12 .5 g of wheat flour at 40°C for 2 hr. The pretreated wheat starch was hy­drolysed with 2 ml glucoamylase (600 AMG units) at 40°C for 4hr. The pretreated hydrolysed wheat starch was fermented by inoculating with the yeast culture @ 1 5% which was found to be optimum. Fermenta­tion was carried out at 30°C in BOD incubator and ethanol produced was estimated.

Identification of yeast-Identification of yeast strains was based on morphological , physiological and biochemical tests by standard procedures l2 .

Results and Discussion Yeast isolates-Out of 3 1 samples used for en­

richment and isolation, yeast strains were obtained from 1 5 samples and given code numbers from SM- l to SM- 1 5 (Table 1 ). Further selection was based on efficient starch fermenting ability and high glucoamy­lase activity.

Growth, glucoamylase activity and ethanol produc­tion of different strains in SYP broth-The growth of different yeast strains was studied by inoculating equal biomass into 1 00 ml of SYP broth containing 2% starch. Growth was estimated in terms of O.D. after incubation at 30°C under shaking conditions. It

Table 1 -Isolation of amylolytic yeasts from different sources

Sources No. of Samples Code No. of strain Colony Microscopic samples showing morphology observation

growth "

Flower 4 2 SM- l , SM-2 White Round cells Honey 2 1 SM-3 Creamy Oval cells Molasses 2 2 SM-4, SM- 1 5 White, Creamy Round cells Bakery dough 2 2 SM-5, SM-6 Creamy, White Round cells Wheat flour 3 2 SM-8, SM- 1 3 White, Creamy Round cells Rice flour dough 2 2 SM-7, SM- 14 White Round cells Fruits 4 2 SM-9, SM- l O White Round cells Soil 4 2 SM- l l , SM- 12 White, Creamy Round cells Ceral based 7 Nil fermented food

... ,

SHARMA et al.: FERMENTATION OF STARCH TO ETHANOL BY AN AMYLOLYTIC YEAST SM- IO 327

was found that different yeast strains differed with respect to their growth in SYP medium (Table 2). Better growth in terms of 0.0. in the range of 2.300 to 2.900 was observed in case of strains SM-3, SM- l O and SM- 14 only . In comparison, strains SM- l , SM-8, SM- 15 attained a maximum 0.0. only in the range of 0.62-0.90 under similar conditions. Very poor growth was seen in rest of the strains.

All the fifteen strains (SM- l to SM- 15 ) were screened for glucoamylase activity. Only three strains SM-3, SM- l O and SM- 14 exhibited higher glucoamy­lase activity of 49, 80 and 22 units mr' , respectively. In other strains very low glucoamylase activity ( 10 units mr l or less) was observed (Table 2).

Ethanol production by different yeast strains when tested in SFM medium containing 10% starch with equal inocula, strain SM- l O produced maximum amount of 3.5% ethanol after 72 hr while strains SM-3 and SM-14 produced only 1 .5% and 2.5% under simi­lar conditions. Other strains were found to produce less than 1 % ethanol.

The amount of ethanol produced by different yeast strains in fermentation medium containing 15% dex­trose was studied by inoculating equal biomass @ 0.5g per 100 rnl (wet weight). Three strains SM-3, SM- l O and SM- 14 were able to produce a maximum of 5 .0, 6.5 and 6.0% ethanol, respectively after 72 hr which was much higher than the amount of ethanol produced by these strains in starch medium. However, yeast strain SM- l O was found to be better with respect to growth in starch, glucoamylase activity and ethanol

production from starch as well as dextrose (Table 2). Hence this strain was used further for ethanol produc­tion from wheat flour.

Ethanol production by yeast strain SM-lO from wheat flour-Wheat flour slurry obtained after ge­latinization by boiling for 5 min on a magnetic stirrer was inoculated at different inoculum levels @ 15 , 30 and 45%. Fermentation was carried out at 30°C and amount of ethanol produced was estimated. It was found that yeast strain SM- l O produced only 1 .75% ethanol using 15% inoculum which was increased to 2.25% by using inoculum at the level of 45% (Table 3).

Ethanol production by yeast strain SM-lO from wheat flour using commercial enzymes- Wheat flour hydrolysate was treated with different concentrations of commercial enzymes pepsin, diastase and glucoa­mylase as shown in Fig. 1 . Wheat flour hydrolysate was inoculated at the level of 1 5 % with an actively growing yeast inoculum of SM- lO. Fermentation was carried out at 30°C and ethanol produced was esti­mated. It was found that treatment of wheat hydrolys­ate with commercial enzymes increased the ethanol production.

Table 3-Ethanol production by yeast strain SM-IO from wheat flour

Inoculum (%)

1 5 30 45

24 hr 0.25 0.48 0.53

Ethanol (% v/v) 48 hr 0.75 1 . 10 1 .23

72 hr 1 .75 2. 1 0 2.25

Table 2-Screening of yeast strains for growth, glucoamylase activity and ethanol production

Yeast Growth 0.0. Glucoamylase Ethanol Eroduction (%v/v) from activity units mr l Starch Dextrose

SM-l 0.909 4.75 0.25 0.2 SM-2 0.542 1 0.00 0.00 0.0 SM-3 2.300 49.00 1 .50 5.0 SM-4 0.830 1 0.00 0.25 0.6 SM-5 0.720 5.00 0.50 0.7 SM-6 0.5 1 0 8.25 0.25 0.6 SM-7 0.895 1 0.00 0.00 0.5 SM-8 0,620 5.75 0.40 0.5 SM-9 0.607 5.25 0.50 0.7 SM- IO 2,600 80.00 3.50 6.5 SM- l l 0,430 4.25 0.00 0.0 SM- 1 2 0.670 0.00 0.00 0.0 SM- 1 3 0,280 3.00 0.25 0,5 SM- 14 2.900 22.00 2.50 6.0 SM- 1 5 0.904 0.00 0.00 0.5

Growth, glucoamylase activity and ethanol production were estimated after 72 hr

328 INDIAN J EXP BIOL, MARCH 2002

Table 4-Ethanol production from pretreated wheat flour by yeast strain SM- IO after treatment with different concentrations of

commercial enzymes

Conc. of enzymes

A B

24 hr

1 .25 2.00

Ethanol (% v/v) 48 hr

2.60 3.75

72 hr

4.25 5.20

A = Pepsin 13 .5 mg (40 units), diastase 1 .3 mg ( 1 690 units) glu­coamylase 2 ml (600 AMG units)

B = Pepsin 27.0 mg (80 units), diastase 2.6 mg (3380 units) glu­coamylase 4 ml ( 1200 AMG units)

Inoculum level 1 5%

Table 5 -Morphological and biochemical properties of yeast strain SM- I O

Shape of the cells Vegetative reproduction Colour Cell size Formation of ascospore Assimilation of nitrogen

Nitrate Nitrite L-Iysine

Round Budding White

6x4.5 J.1m 1 -4

Table 6-Assimilation and fermentation of different sugars by yeast strain SM- I O

Sugar Assimilation of sugar Fermentation of su'gar

Maltose Galactose Sorbose Cellobiose Trehalose Lactose Melibiose Raffinose Insulin Xylose Rhamnose Sucrose Dextrose

+ +

+ +

+ +

+ +

+ +

By using pepsin, diastase and glucoamylase ( 1 3 .5 mg, 1 .3 mg and 2 ml, respectively) maximum of 4.25% ethanol was produced after 72 hr using 1 5% inoculum. Further, by increasing the concentration of enzymes (27.0 mg pepsin, 2.6 mg diastase and 4 ml glucoamylase) a maximum of 5 .2% ethanol was pro­duced by strain SM- 1O by using same level of inocu­lum (Table 4).

Identification of yeast strain SM-IO -Various morphological, physiological and biochemical charac­teristics of yeast strain SM-1 O confirm well with the characteristics of Saccharomyces diastaticus and hence this strain was identified as S. diastaticus SM-10 (Tables 5 and 6).

Thus, after a thorough screening of various amy­lolytic yeasts isolated from different sources an amy­lolytic yeast S. diastaticus SM- 1O was obtained. This strain had the ability to assimilate and ferment starch as well as alcohol production from starch. Further work on standardization of conditions for high glu­coamylase activity as well as characterization and purification of glucoamylase is continued.

References 1 Abourzied M M & Reddy C A, Fermentation of starch to

ethanol by a complementary mixture of an amylolytic yeast and Saccharomyces cerevisiae. Biotechnol Lett, 9 ( 1 987) 59.

2 Abourzied M M & Reddy C A, Direct fermentation of potato starch to ethanol by coculture of Aspergillus niger and sac­charomyces cerevisiae, Appl Environ Microbiol, 52 ( 1986) 1055.

3 Elegado F B, Mendoza E R & Pham C B, Simultaneous sac­charification and fermentation of raw starch with ethanol stripping and rectification, Philip J Sci. 1 19 (3) ( 1990) 205.

4 Ueda S. Zenin C T. Monteiro D A & Pork Y K, Production of ethanol from raw cassava starch by a non-conventional fermentation method. Biotechnol Bioeng. 23 ( 198 1 ) 29 1 .

5 Chi Z M & Liu Z R, High concentration ethanol fermentation of raw com starch by Saccharomyces sp. H.Q. Chinese J Biotech, 1 0 ( 1 994) 1 30.

6 Sakai T K-Ikoo, Satioh K & Karturogi T, Use of protoplast fusion for the development of rapid starch fermenting strain of Saccharomyces diastaticus. Agri bioi Chem. 50 ( 1986) 297.

7 Sharma S & Tauro P, Detection and biochemical characteri­zation of fast and slow ethanol producing yeasts, Mircen J Appl Microbiol Biotechnol 3 ( 1 987) 1 55.

8 Caputi A, Ueda J M & Brown T, Spectrophotometric deter­mination of chromic complex formed during oxidation of al­cohol, Am J Enol Viticult, 1 9 ( 1 968) 1 60.

9 Lemmel S A, Himsch R C & Korus R A, Kinetics of growth and amylase production of Saccharomycopsis fibuligera on potato processing waste water, Appl Environ Microbiol, 39 ( 1 980) 387.

10 Dabas R, Ethanol production from wheat, M.Sc. Thesis CCS Haryana Agric Univ, Hisar, 1 996.

1 1 De Menezes T J B, Starchy materials for alcohol fuel produc­tion, Process Biochem. 1 7 ( 1989) 32.

12 Kreger-van Rij N J W, in The yeast a taxonomic study, 3rd ed (Elsevier Publication Amesterdam) 1 984.