Bioresource Technology 83 (2002) 199202

Eect of UV radiation on thermotolerance, ethanol tolerance and

osmotolerance of Saccharomyces cerevisiae VS1 and VS3 strains
M. Sridhar, N. Kiran Sree, L. Venkateswar Rao*
Department of Microbiology, Osmania University, Hyderabad 500 007, India
Received 19 September 2001; received in revised form 15 November 2001; accepted 23 November 2001

Abstract
After a previous mass screening and enrichment programme for the isolation of thermotolerant yeasts, VS1 , VS2 , VS3 and VS4
strains isolated from soil samples, collected within the hot regions of Kothagudem Thermal Power Plant, AP, India, had a better
thermotolerance, osmotolerance and ethanol tolerance than the other isolates. Among these isolates VS1 and VS3 were best performers. Eorts were made to further improve their osmotolerance, thermotolerance and ethanol tolerance by treating them with
UV radiation. Mutants of VS1 and VS3 produced more biomass and ethanol than the parent strains at high temperature and glucose
concentrations. The amount of biomass produced by VS1 and VS3 mutants was 0.25 and 0:20 g l
1 more than the parent strains at
42 C using 2% glucose. At high glucose concentrations VS1 and VS3 mutants produced biomass which was 0.70 and 0:30 g l
1 at 30
C and 0.10 and 0:20 g l
1 at 40 C more than the parent strains. The amount of ethanol produced by the mutants (VS1 and VS3 )
was 8.20 and 1:20 g l
1 more than the parent strains at 42 C using 150 g l
1 glucose. More ethanol was produced by mutants (VS1
and VS3 ) than the parents at high glucose concentrations of 5.0 and 6:0 g l
1 at 30 C and 13.0 and 3:0 g l
1 at 42 C, respectively.
These results indicated that UV mutagenesis can be used for improving thermotolerance, ethanol tolerance and osmotolerance in
VS1 and VS3 yeast strains. 2002 Elsevier Science Ltd. All rights reserved.
Keywords: Thermotolerance; Osmotolerance; Saccharomyces cerevisiae; Ethanol tolerance; UV radiation

1. Introduction
Thermotolerant yeasts help in reducing cooling costs,
distillation costs and have faster fermentation rates and
also help in decreasing contamination chances during
fermentation (Banat et al., 1996; Gera et al., 1997; Dalel
Singh et al., 1998; Kiran Sree et al., 1999).
The world of fermentation ethanol industry is very
large, therefore any small improvement in the eciency
of ethanol production by improved thermotolerant
yeasts could be economically signicant. Yeast strains
normally used in industrial processes have limited osmotolerance and thermotolerance. Due to this, alcohol
fermentations are carried out at comparatively low
temperature and low sugar concentrations (Salmon and
Mauricio, 1994). Osmotolerant and ethanol tolerant
yeasts are advantageous in ethanol industry, because
growth and fermentation at high initial sugar concentration not only decreases the chances of contamination
*

Corresponding author. Tel.: +91-40-709-0661, +91-40-7098951x246; fax: +91-40-709-9020.

E-mail address: vrlinga@yahoo.com (L. Venkateswar Rao).

but also helps in obtaining high ethanol concentrations,

which decrease the distillation costs (Bertolini et al.,
1991; Kiran Sree et al., 2000a,b).
Induced mutagenesis using physical and chemical
mutagens seems to be a simple and rational approach
for yeast strain improvement. Selection after treatment
with mutagens has been used to a considerable extent
in improvement of yeasts. Demchemko and Kobrina
(1979) examined mutagenesis as a means to generate
thermotolerance in yeasts. Induction of mutation by UV
and MNNG followed by selection was successfully used
for improvement of baking yeast strains in Poland and
USSR (Johnston and Oberman, 1979). Uma and Polasa
(1990) have reported the eect of UV in improving the
biomass and ethanol yield using mesophilic Saccharomyces cerevisiae at 30 C. Kiran Sree et al. (2000a,b)
reported the isolation of thermotolerant osmotolerant
occulating Saccahromyces cerevisiae (VS1 , VS2 , VS3
and VS4 ) strains. Because of the potential advantages of
thermotolerant, ethanol tolerant and osmotolerant
yeasts, the present investigation was aimed at further
improving VS1 and VS3 strains by treatment with UV
radiation.

0960-8524/02/$ - see front matter 2002 Elsevier Science Ltd. All rights reserved.
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M. Sridhar et al. / Bioresource Technology 83 (2002) 199202

2. Methods
Among the four isolates (VS1 , VS2 , VS3 and VS4 ),
isolated from the soil samples collected within the hot
regions of Kothgudem Thermal Power Plant, VS1 and
VS3 strains had better thermotolerance, osmotolerance
and ethanol tolerance compared to other strains tested.
Hence VS1 and VS3 strains were selected for UV
mutagenesis to further improve their thermotolerance,
ethanol tolerance and osmotolerance.

and 350 g l
1 glucose at both 30 and 42 C, respectively.

Ethanol concentrations were determined by using high
pressure gas chromatography (HPGC, Hewlett Packard
4890 D, poropack-Q, mesh size 80100 lm) equipped
with a ame ionization detector. The chromatogram was
run at 160 C oven temperature and 170 C injecting
temperature using nitrogen as carrier gas and hydrogen
as a aming gas both at a ow rate of 32 ml min
1 . All
the experiments were replicated twice and the average
values are presented. A variation of about 5% was seen
between the two experiments.

2.1. Media
2.4. Statistical analysis
VS1 and VS3 strains maintained on YEPD agar were
inoculated into 250 ml conical asks with 100 ml YEPD
medium containing yeast extract 1%, peptone 2%,
dextrose 2%, pH 5.5 and the asks were incubated at
30 C on a shaker at 150 rpm.

The data obtained after mutation experiments were

subjected to statistical analysis using t-test.

3. Results and discussion

2.2. UV mutagenesis
Exponentially growing (16 h) VS1 and VS3 cultures
were washed after centrifugation, re-suspended in 0.1 M
phosphate buer of pH-7.0 and adjusted to 108 cells per
ml by a turbidometric method. About 10 ml of VS1 and
VS3 cultures were transferred to sterile petriplates and
irradiated for 6 min with a UV lamp of 30 W at a distance of 30 cm. UV mutagenesis was carried out according to the method of Vashishat and Beena (1983).
Samples were taken after every 15 s and, after serial
dilution in buer, plated onto YEPD plates and incubated at 30 C.
About 100 colonies from dierent plates exposed to
dierent time intervals were selected, based on colony
morphology and size. Five percent inoculum of 100
colonies, grown in YEPD broth at 30 C was transferred
to 250 ml conical asks containing 100 ml of YEPD
medium. These asks were incubated at 42 C for
selecting the best thermotolerant mutants, capable of
showing good growth at that temperature. The best
performing ve isolates of VS1 and VS3 were tested for
their ethanol production at 30 and 42 C using 150 g l
1
glucose.
2.3. Analytical methods
Thermotolerance of the mutants was tested using 2%
glucose by estimating the biomass yield at 42 C. For
estimation of biomass the cells were centrifuged and the
wet weight of the pellet was taken. Ethanol yield of the
thermotolerant mutants was tested at 30 and 42 C using
150 g l
1 glucose. Osmotolerance of the mutants was
tested by estimating the amount of biomass produced
using 150, 200, 250, 300 and 350 g l
1 glucose at both 30
and 40 C. Ethanol tolerance of the mutants was tested
by estimating the amount of ethanol produced with 250

After UV treatment LD50 was found to be 150 s, and

100% killing of cells was obtained at 5 min. More biomass was produced by VS3 100 and VS1 40 mutants
using 2% glucose. Therefore only VS3 100 and VS1 40
mutants were used in all the experiments. The biomass
produced by VS3 mutant was 0:20 g l
1 1:80 g l
1
more than the parent VS3 strain (1:60 g l
1 ) while the
biomass produced by VS1 mutant was 0:25 g l
1
(1:75 g l
1 ) more than the VS1 parent 1:50 g l
1 using
2% glucose. These results indicated that UV mutagenesis
was eective in improving the biomass yield of VS1 and
VS3 strains which was statistically signicant as shown
in Table 3.
After testing the biomass yield, these mutants were
tested for their ethanol production at 30 and 42 C using
150 g l
1 glucose. The amount of ethanol produced by
UV-treated VS1 40 mutant increased to 8:2 g l
1
(58:2 g l
1 ) more than the parent VS1 50:0 g l
1 . While
the amount of ethanol produced by UV-treated VS3 100
mutant increased by only 1:2 g l
1 (59:2 g l
1 ) compared to that of parent VS3 (58:0 g l
1 ) at 42 C, the
amount of ethanol produced by these mutants at 30 C
was the same as that of the parent strains. The improvement in ethanol yield of VS1 strain was statistically
signicant compared to that of VS3 at 42 C.
For isolating the best performing osmotolerant mutants, the inocula from 100 colonies obtained after UV
treatment were inoculated into YEPD medium containing 150, 200, 250, 300 and 350 g l
1 glucose and
incubated at 30 and 40 C. The biomass and ethanol
yield of parent, mutated VS1 and VS3 strains using different glucose concentrations at 30 and 40 C are shown
in Table 1. It was observed that the osmotolerance was
greater in thermotolerant mutants.
The maximum biomass produced by mutant VS1 was
0:60 g l
1 more than the parent VS1 at 30 C. The

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201

Table 1
Biomass of parent and mutated VS1 and VS3 strains using dierent sugar concentrations at 30 and 40 C
Strain

Glucose concentration (g l
1 )

150 BY

VS1 (parent)
VS3 (parent)
UV-VS3 100
UV-VS1 40

200 BY

250 BY

300 BY

350 BY

30 C

40 C

30 C

40 C

30 C

40 C

30 C

40 C

30 C

40 C

1.80
1.90
2.10
2.10

1.00
1.10
1.20
1.10

1.70
1.80
1.90
1.90

0.90
1.00
0.90
0.90

1.60
1.70
1.80
1.90

0.80
0.90
1.00
0.90

1.20
1.50
1.80
1.90

0.80
0.90
0.90
0.90

1.20
1.30
1.50
1.50

0.40
0.60
0.70
0.60

BY Biomass yield in g l
1 .

Table 2
Ethanol yield (EY) of parent and UV-treated VS1 and VS3 mutants at
both 30 and 42 C using 250 and 350 g l
1 glucose
Strain

250 EY

350 EY

30 C

40 C

30 C

40 C

VS1 (parent)
VS3 (parent)
UV-VS3 100
UV-VS1 40

88.0
95.0
98.0
93.0

50.0
61.0
62.0
58.0

83.0
82.0
88.0
86.0

38.0
50.0
53.0
51.0

EY Ethanol yield in g l
1 .

Table 3
Statistical analysis of biomass and ethanol yields of UV-treated VS1
and VS3 mutants using t-test
Sample
Biomass
UV-VS1 on
YEPD at 42 C
UV-VS3 on
YEPD at 42 C
Ethanol
UV-VS1
UV-VS3

t-Value
(0.05%)

t-c Calculated value

increased by 8:0 g l
1 using 250 g l
1 glucose and

13:0 g l
1 using 350 g l
1 glucose. These results indicate
that UV rays are eective in improving the ethanol
tolerance of yeast strains at 30 and 42 C.
The amount of ethanol produced by mutant VS3 was
increased by 3.0 and 1:0 g l
1 using 250 g l
1 glucose
and 6.0 and 3:0 g l
1 using 350 g l
1 glucose at 30 and
42 C, respectively. The improvement of ethanol tolerance of VS1 was statistically signicant at both 30 and
42 C whereas VS3 mutant was statistically signicant
only at 30 C using 250 and 350 g l
1 glucose.

4. Conclusion

Signicance

2.776

4.724

2.776

4.652

2.776
2.776

13.80
0.7507

+
)

The results obtained indicate that UV-treated mutants of VS1 and VS3 acquired more osmotolerance,
thermotolerance and ethanol tolerance than their parent
counterparts and these mutants will be more useful for
ethanol production.

Acknowledgements

(+): Statistically signicant; ()): Statistically not signicant.

We are thankful to University Grants Commission,

New Delhi for a grant-in-aid during this investigation.
maximum amount of biomass produced by UV-treated
VS3 mutant was 0:40 g l
1 more than the parent VS3
strain at 30 C. The biomass yield of VS3 and VS1 mutants remained the same as that of the parent strains at
40 C. This may be due to the combined eect of high
temperature and high initial sugar concentration on the
growth of these organisms. These results indicate that
UV mutagenesis was eective in improving the osmotolerance at 30 C.
These mutants were also tested for their ethanol tolerance at 30 and 42 C using 250 and 350 g l
1 glucose.
The ethanol yields of parents and mutant VS1 and VS3
strains are shown in Table 2. VS1 mutant produced
more ethanol than the parent VS1 at both 30 and 42 C
using 250 and 350 g l
1 glucose. The amount of ethanol
produced by mutant VS1 increased by 5:0 g l
1 using
250 g l
1 glucose and 3:0 g l
1 using 350 g l
1 glucose at
30 C, while the amount of ethanol produced at 42 C

References
Banat, I.M., Singh, D., Marchant, R., 1996. The use of thermotolerant
fermentative Kluyveromyces marxianus IMB 3 yeast strain for
ethanol production. Acta Biotechnol. 16, 215223.
Bertolini, M.C., Ernandes, J.R., Laluce, C., 1991. New yeast strains for
alcoholic fermentation at higher sugar concentration. Biotechnol.
Lett. 13, 197202.
Dalel Singh, Banat, I.M., Nigam, P., Merchant, R., 1998. Industrial
scale ethanol production using the thermotolerant yeast Kluyveromyces marxianus IMB 3 in an Indian distillery. Biotechnol. Lett.
20, 753755.
Demchemko, L.N., Kobrina, Y.P., 1979. Preparation of thermostable
mutants of bakers yeast by induced mutagenesis. Microbiology 48,
730734.
Gera, R., Dhamija, S.S., Gera, T., Dalel Singh, 1997. Intergenic
ethanol producing hybrids of thermotolerant Kluyveromyces and
nonthermotolerant Saccharomyces cerevisiae. Biotechnol. Lett. 19,
189193.

202

M. Sridhar et al. / Bioresource Technology 83 (2002) 199202

Johnston, J.R., Oberman, H., 1979. Yeast genetics in industry. Prog.

Ind. Microbiol. 15, 151155.
Kiran Sree, N., Sridhar, M., Venkateswar Rao, L., Ashok Pandey,
1999. Ethanol production in solid substrate fermentation using
thermotolerant yeasts. Process Biochem. 34, 115119.
Kiran Sree, N., Sridhar, M., Suresh, K., Banat, I.M., Venkateswar
Rao, L., 2000a. High alcohol production by repeated batch
fermentation using immobilized, osmotolerant Saccharomyces
cerevisiae. J. Ind. Microbiol. Biotechnol. 24, 222226.
Kiran Sree, N., Sridhar, M., Suresh, K., Banat, I.M., Venkateswar
Rao, L., 2000b. Isolation of thermotolerant, osmotolerant, occu-

lating, Saccharomyces cerevisiae for ethanol production. Bioresource Technol. 72, 4346.
Salmon, J.M., Mauricio, J.C., 1994. Relationship between sugar
uptake kinetics and total sugar consumption in dierent Industrial
Saccharomyces cerevisiae strains during alcohol fermentation.
Biotechnol. Lett. 16, 8994.
Uma, V., Polasa, H., 1990. Enhanced ethanol production by using
mutagens. Microbiol. Biotechnol. 5, 14.
Vashishat, R.K., Beena, K., 1983. Eect of colchicine and methylated
purines on UV-induced gene conversion in Saccharomyces cerevisiae. Indian J. Exp. Biol. 21, 367.