Current Research in Microbiology and Biotechnology

Vol. 2, No. 1 (2014): 316-324

Research Article
Open Access
ISSN: 2320-2246

Screening, identification and characterization of

alcohol tolerant potential bioethanol producing
yeasts
Mir Naiman Ali* and Mohammed Mazharuddin Khan
Department of Microbiology, Mumtaz Degree and P.G College, Hyderabad, India

* Corresponding author: Mir Naiman Ali, email: naimanali@gmail.com

Received: 28 December 2013 Accepted: 08 January 2013 Online: 15 January 2014

ABSTRACT
In the present study fourteen cultures were isolated from soil samples, fruits and fermented products, of which
seven were found to be yeasts. These seven yeasts were subjected to thorough identification scheme upto
species level by cultural, morphological, microscopic, biochemical and physiological studies and by comparing
the results with reference strain Saccharomyces cerevisiae MTCC 170. The yeasts were characterized with respect
to temperature tolerance, ethanol tolerance and osmotolerance. Batch stationary fermentation was carried out in
Erlenmeyer flasks for bioethanol production. Growth and fermentation kinetics were calculated for stationary
fermentation. Three species (TA, C2 and K2) were identified as Saccharomyces cerevisiae, two (S1 and S2) were
identified as Saccharomyces rosinii, and the other two (K1 and S3) were identified as Saccharomyces exiguus and
Rhodotorula minuta respectively. Among all yeasts Saccharomyces cerevisiae TA and C2 strains were high ethanol
tolerant (tolerated 14% ethanol), high osmotolerant (tolerated 20% sugar) and high bioethanol producing
strains with a yield of 32 and 28 g/l for TA and C2 respectively. Results indicated that fermentation kinetics with
S. cerevisiae TA and C2 strains were faster than other yeast strains including the reference strain MTCC 170 with
the ethanol yield (Yp/s= 0.160 and 0.155 g g-1), volumetric substrate uptake (QS= 2.638 and 2.50 g L-1 h-1),
conversion rate into ethanol (16.80 and 15.50 %) and volumetric product productivity (Qp,= 0.44 and 0.38 g L-1 h-
1) respectively.

Keywords: Saccharomyces; bioethanol; stationary fermentation; fermentation kinetics

INTRODUCTION
Yeasts are wide spread in terrestrial, aquatic and aerial
environments. They have been isolated from natural
substances like leaves, flowers, sweet fruits, grains,
fleshy fungi, exudates of trees, insect, dung and soil [1].
Preferred habitats are plant tissues, leaves and flowers,
fruits, fermented products, soil and salt water.
Members of Saccharomycetales occur with regularity in
the bark of certain deciduous trees [2] and
intermittently in fermenting fruit and other high sugar
environments such as nectar and sap fluxes. Different
methods for isolating and characterizing yeasts from
environmental samples have been described by Phaff et
al., [3] and Spencer and Spencer [1].
Various yeast species found on the grape and on winery
surfaces participate in spontaneous fermentations.
Yeasts of the genera Kloeckera, Hanseniaspora and
Candida predominate in the early stages of the
fermentation, followed by several spp. of
Matschnikowia and Pichia in the middle stages, when
the ethanol rises to 3-4 % [4]. The latter stages of
spontaneous wine fermentation invariably are
dominated by the alcohol-tolerant strains of the
Saccharomyces sensu strict group of yeasts. This taxon
consists of four yeast species, namely Saccharomyces
bayanus, Saccharomyces cerevisiae, Saccharomyces
paradoxus and Saccharomyces pastorians.
The population of microflora on the substrate always
depends on the pH of the substrate. Since fruits are
acidic in nature they are predominantly inhabited by
yeasts [5]. Yeast strains associated with fruit surfaces
are capable of converting wide range of sugars into
alcohol and they can also tolerate high concentration of
alcohol. In assessing a yeast strain for industrial use,
specific physiological properties are required. [6].

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Ethanol tolerance, sugar tolerance and invertase
activities are some of the important properties for use
in industrial ethanol production [7]. The efficiency of
yeast strains is determined by their ability to utilize
sugar substances, ethanol tolerance capacity, growth at
370C and alcohol production capacity of yeast strains
[8].
Organisms generally employed for bioethanol
production are strains of yeast Saccharomyces
cerevisiae and bacteria Zymmomonas mobilis. But S.
cerevisiae is commonly employed for bioconversion of
substrate to the higher yield of bioethanol under
controlled optimization parameters [9]. S.cerevisiae is
also reported as the most studied and biochemically
best understood species of the yeast domain. It is best
known for its domesticated role in the production of
fermented products. This yeast converts hexose sugars
to ethanol, CO2, and a variety of compounds including
alcohols, esters, aldehydes and acids that contribute to
the sensory attributes of the food and beverage [10].
Fermentation of carbohydrates in fruits, grains and
other biomass to ethanol by S. cerevisiae is the critical
process for a wide range of products from fine wines to
gasoline additives [11]. Keeping in view the importance
of the yeast S. cerevisiae and its proved higher ethanol
Table 1. Details of sample collection for isolation of yeasts
S.No Source sample Sample Type
Sugarcane market (Nampally)
1. Soil samples Vineyard (Shamshabad)
Fruit Market (Kothapet)
2. Fruit samples Grapes
Tamarind
3. Fermented products Yoghurt
Kanji
Isolation of yeasts
i) Soil Samples: One gram of each soil sample was
suspended in 9 ml of sterile deionized water and
treated according to the technique of Diriye et al., [12].
0.1ml of each suspension was plated by spread plate
method onto agarized YEPD+ C (Yeast extract peptone
Glucose + Chloramphenicol) media containing: Yeast
extract, 3.0g; peptone, 10.0g; glucose, 10.0g;
chloramphenicol, 0.03g; distilled water, 1000ml; PH,
5.5; and incubated for 3 days at 300C for isolation of
Yeast.
ii) Fruit Samples: Grapes (5) and tamarind (20g) were
washed with sterile distilled water and washed water
was plated onto YEPD+C medium. Grapes (2) were also
rolled on the surface of YEPD+C agar plates to collect
superficially attached yeast strains. After inoculation
the plates were incubated at 300C for 3 days.
iii) Fermented products: Yoghurt and Kanji (Kanji is a
liquid generated by the village people by boiling rice
and fermented for three days with indigenous
microorganisms) samples were serially diluted 10 fold
and 0.1 of each sample was inoculated on to YEPG+C
medium. The plates were incubated at 300C for 3 days.
After 3 days of incubation plates were examined for
colony formation and colonies suggestive of yeasts
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production ability the present study was undertaken
with following objectives- a) to screen and identify
potent bioethanol producers from natural sources like
soil, fruits and fermented products, b) to characterize
them for ethanol tolerance, sugar tolerance and
osmotolerance and c) to perform batch fermentation
for testing the higher ethanol production ability of
isolated yeast strains.
MATERIALS AND METHODS
Screening of yeasts
For screening of yeasts three different types of samples
were selected- soil samples, fruit samples and
fermented products. a) Soil samples were collected
from 3 different locations from Hyderabad, Metropolis
and nearby districts. b) Fruit samples were collected
from a local fruit market in Hyderabad. c) Fermented
products were collected from a well known
supermarket in Hyderabad and from Khammam
district of Andhra Pradesh, India. The details of type of
sample and location are shown in Table-1 below. All
samples are collected in sterile containers, transported
to the laboratory and kept refrigerated until further
processing.
Location
Centrally located in Hyderabad City
Located 30 km from Hyderabad, City
Centrally located in Hyderabad City
Supermarket in Hyderabad
Supermarket in Hyderabad
Supermarket in Hyderabad
Khamman District of Andhra Pradesh
were preliminary identified by microscopic
examination of smears for studying cell morphology
and budding characteristics. For obtaining pure culture
loop full of colonies from above plates were inoculated
in YEPD broth and also 0.1 ml of each culture was
streaked onto YEPD agar plates. The plates were
incubated at 300C for 3 days.
Identification of yeasts
The isolated yeast strains were identified by studying
specific morphological, biochemical and physiological
characteristics as given by Kurtzman and Fell [13]. For
comparison reference strain of Saccharomyces
cerevisiae MTCC 170 procured from MTCC (Microbial
Type Culture Collection Centre and Gene Bank)
Chandigarh, India was used in all experiments for
identification and characterization of yeasts isolates.
Identification scheme: To identify yeast, the following
scheme given by Martini and Martini [14] was adopted:
colony colour, shape and texture were examined. If the
colony is black to brown in colour or moist mycelial in
texture, a direct mount is prepared. If the colony is pink
to red, the colony is streaked for isolation. After
incubation, the presence of satellite colonies is checked.
Colony is examined microscopically for forcibly
discharged conidia. Using the flow chart genus is
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determined based on morphology. Assimilations will be
necessary to determine the species. If the colony is
white or cream coloured, germ tube test is performed.
Negative germ tube test confirms the purity of yeast
isolates which will be further tested by performing
following tests for identification upto species level.
Growth in 5% malt extract: Isolates were inoculated
in 5% malt extract broth and incubated at 250C for 3
days. After 3 days cells were microscopically examined
for studying morphology (bud formation).
Formation of ascospores: Ascospore formation was
studied on acetate agar (2.5% yeast extract, 1%
dextrose, 10% potassium acetate, agar 3% and PH 6.5).
Isolates were inoculated in autoclaved acetate agar and
incubated at 250C for 6 days. After 6 days cells were
examined microscopically under 40 x and 100 x
objectives for formation of ascospores.
Assimilation of various Carbon Compounds:
Fermentation was carried out in test tubes with
durham tube inserted. In each test tube, Yeast extract
(1%), peptone (2%) and 2% weight by volume of single
carbon source was taken. The carbon compounds
which were included in the system are Glucose,
Galactose, L-Sorbose, D-Ribose, D-Xylose, L-Arabinose,
Rhamnose, -Methylglucoside, Sucrose, Maltose and
Trehalose. Amino sugars: D-Glucosamine, N-acetyl-D-
Glucosamine were also used. Tubes were inoculated
with yeast isolates and incubated at 250C until gas is
visible in the inserted durham tube and if gas was not
produced, fermentation was carried up to 28 days. The
results indicated fermentation by gas collecting in
durhams tube or assimilation by turbidity only.
Assimilation of Nitrogen Compounds: Potassium
nitrate (40 mM) is dissolved in Difco Yeast Carbon
Base. 2.5 ml of the broth was dispensed in culture tubes
(15 cm, 16 mm width) and was sterilized at 1200C for
20 min. (Ethylamine hydrochloride should not be
sterilized in the presence of glucose). A separately
sterilized concentrated solution was added aseptically
to culture tubes with 2.5 ml sterile Yeast Carbon Base,
to a concentration of 40 mM. The culture tubes were
inoculated with a drop of young culture in Yeast Carbon
Base with 40 mM ammonium chloride (sterilized
separately). For comparison a culture tube with Yeast
Carbon Base without nitrogen source is also inoculated.
All tubes were incubated at 250C in an orbital shaking
incubator up to two weeks and were inspected daily for
growth. Positive growth responses were confirmed by
transferring a loop of culture to the same type of
growth medium which has also shown growth.
The other tests which were carried out as per the
identification scheme are: test for urease (as per
method of Deak and Beuchat [15], tolerance to 1%
acetic acid (only to discriminate Zygosaccharomyces
spp.), resistance to 0.01% cycloheximide and vitamin
requirement (biotin, thiamine and Ca-pantothenate).
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Temperature tolerance
The isolated yeast strains and reference strain
Saccharomyces cerevisiae MTCC 170 were streaked
onto agar plates containing YEPD agar for checking the
effect of temperature on the growth. The plates were
incubated at 25, 28, 30, 35, 37, 40, 42 and 450C for 48
hrs. After 48 hrs plates were observed for colony
formation and the extent of growth was recorded.
Ethanol tolerance
Ethanol tolerance of yeast strains was tested by
inoculating 5% broth culture of each strain in
Erlenmeyer flasks with YEPD broth containing 1% to
18% alcohol (v/v) in triplicates. After inoculation,
flasks were incubated at 300C for 48 hrs. Samples were
taken every 24 hrs and optical density was recorded at
600 nm on U.V-Visible Spectrophotometer (ELICO,
India). All experiments were carried out in triplicates
and mean values were considered.
Sugar tolerance (Osmotolerance)
The yeast strains were tested for their osmotolerance
by testing their growth in YEPD broth (in triplicates)
containing 10, 15, 20, 25, 30 and 35% glucose
concentration. Actively growing yeast cultures (10%)
were inoculated in triplicates and flasks were
incubated for 48 hrs at 300C. Samples were taken every
24 hrs and optical density was recorded at 600 nm.
Bioethanol production
Yeast strains were tested for their bioethanol
production efficiency in 500 ml Erlenmeyer flasks (in
triplicates) containing 200 ml of YEPD broth with 10%,
15% and 20% glucose concentration according to
optimum sugar concentration required by respective
strains. Flasks were incubated at 300C for 72 hrs,
samples were withdrawn every 24 hr for estimation of
bioethanol produced and left over sugar. The
estimation of left over sugar was based on the
dinitrosalicylic acid (DNS) method [16]. A double beam
UV/Visible spectrophotometer was used for measuring
absorbance at 575 nm. Ethanol was determined with
good precision by oxidation with acid dichromate
solution [17] and absorbance was measured at 660 nm.
The fermentation kinetics was studied as per the
formulae given by Bailey and Ollis [18].
RESULTS AND DISCUSSION
Yeast Isolation
In the present study yeast cultures were screened from
soil, fruit samples and fermented products as
mentioned in materials and methods. A total of
fourteen (14) cultures were isolated from these
samples out of which seven were identified as yeasts.
The cultures were identified as yeasts based on colony
characters, microscopic examination and budding
formation. Based on differences in colony morphology,
colony size and microscopic examination these strains
were designated as S1, S2 and S3 (for soil), TA (for
tamarind), C2 (for yoghurt) and K1 & K2 (for kanji).
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Table 2. Colony Characteristics of Yeast isolates

YEAST COLONY COLOR COLONY NATURE APPEARANCE ELEVATION MARGIN COLONY
ISOLATE SIZE (mm)
S1 Light cream Smooth Elongate Convex Entire 4.0 x 15.0
colored
S2 Light Cream Smooth Ovoid Raised Enrire 3.8 x 16.0
colored
S3 Pink colored Smooth Globose Convex Entire 4.5 x 16.5
TA Cream colored Smooth Yeast like Convex Entire 4.0 x 16.5
C2 Cream colored Smooth Glaborous and Yeast Convex Entire 5.0 x 15.0
like
K1 Whitish colored Smooth & shiny Ovoid Concave Entire 5.0 x 12.0
K2 Whitish cream Smooth Yeast like Raised Wavy 6.0 x 13.0
colored
MTCC 170 Cream colored Smooth Yeast like Convex Entire 5.0 x 15.5

Yeast Identification surrounding cycloheximide discs. S1, S2 and K1 strains

These seven yeasts were further used for identification
studies. Colonies formed by yeast isolates were round,
smooth and cream, white to whitish cream colored.
Colony size ranged from 3.8 x 16.0 to 6.0 x 13.0 as
shown in Table-2. Individual cells were oval, elongate,
ovoid to spherical when young and hexagonal when
aged. Cells showed oval, globose, spherical and
ellipsoidal budding. S3, TA, C2, K2 and MTCC 170
strains were sensitive to cycloheximide which was
indicated by formation of a clear inhibitory zone
were resistant to cycloheximide which was confirmed
by growth in presence of cycloheximide discs. All
strains were non-tolerant to acetic acid as shown in
Table-3. All strains did not show any pellicle formation
but sedimentation was observed for TA, C2 and
K1strains. The ascospore formation was observed on
acetate agar. Upon microscopic observation globose,
spherical, round and ellipsoidal ascospores were
observed with 1 to 4 ascospores per ascus.

Table 3. Growth characteristics of Yeast isolates

YEAST BUDDING GROWTH ON PELLICLE FORMATION TOLERANCE TO 1% ASCOSPORES
ISOLATE CHARACTER CYLOHEXIMIDE ACETIC ACID SHAPE NUMBER

S1 Globose Growth observed No pellicle observed Non tolerant Round 2

S2 Globose Growth observed No pellicle observed Non tolerant Globose 2

S3 Ovoidal No growth No pellicle observed Non tolerant Globose 3

TA Globose No growth No pellicle observed but Non tolerant Globose 4

Sedimentation in YEPD
broth
C2 Ellipsoidal No growth No pellicle observed but Non tolerant Globose 4
Sedimentation in YEPD
broth
K1 Spherical Growth observed No pellicle observed but Non tolerant Spherical 3
Sedimentation in YEPD
broth
K2 Oval shaped No growth No pellicle observed Non tolerant Globose 4

MTCC 170 Globose No growth No pellicle observed Non tolerant Globose 4

Morphological, biological and physiological studies
were carried out as given by Martini and Martini [14]
and species was identified as given by Barnett et al.,
[19] and Kurtzman et al., [13]. Saccharomyces cerevisiae
MTCC 170 was also used as a reference strain to
compare and identify isolated yeast strains. Bud
formation was observed for all strains (Table-4).
Glucose and sucrose were fermented and assimilated
by all strains except S1 and S2 strains. TA, K1 and
MTCC 170 strains fermented and assimilated trehalose.
TA and MTCC 170 also fermented soluble starch.
Galactose was assimilated and fermented by all strains
except C2 and K2. Maltose was fermented by TA, C2 and
MTCC 170 strains. C2, K1 and K2 strains fermented
raffinose. Delayed fermentation of mannitol was
observed for C2 and MTCC 170 strains. Melibiose
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fermentation was observed only with TA strain. Ribose
and xylose were fermented and assimilated only by S3
strain.
All seven strains did not show, true mycelium,
fragmentation and no pellicle formation was observed.
Sorbose, arabinose, rhamnose, gluosamine, erythriol,
inositol, glucuronate and 2-keto-D-gluconate were not
fermented and assimilated by all strains. Potassium
nitrate and ethylamine were not assimilated by any
strain. Except S3 strain (urease +) all strains were
scored as urease negative.
After studying morphological, biological and
physiological characteristics it was confirmed that
isolated yeast strains belong to four species of yeasts.
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S1 and S2 strains were identified as Saccharomyces
rosinii, S3 strain was identified as Rhodotorula minuta,
K1strain was identified as Saccharomyces exiguus and
TA, C2 and K2 were identified as Saccharomyces
cerevisiae. The confirmation of identification was made
after comparison with standard strain of MTCC 170 by
adopting identification scheme. In another study
Moneke AN, et al., [20] isolated six morphologically
different yeast strains from orchard soil of Nsukka,
Nigeria, which were identified as Saccharomyces yeasts
by adopting similar methodology. Similar isolation and
identification of yeasts was carried out by Brooks AA,
[21] in which eight yeasts were isolated from ripe
banana peels and five were subjected to identification
studies. Three out of five were identified as
Saccharomyces cerevisiae, the other two were
identified as Debaromyces hansenii and Saccharomyces
kluyveri respectively.

Table 4. Morphological, Biochemical and Physiological characteristics of Yeast Isolates

IDENTITY OF YEAST ISOLATES

Rhodotorula minuta

cerevsiae MTCC 170

Saccharomyces

Saccharomyces

Saccharomyces

Saccharomyces

Saccharomyces

Saccharomyces

Saccharomyces
CHARACTERISTICS

cerevisiae TA

cerevisiae K2
cerevisiae C2

exiguus K1
rosinii S1

rosinii S2

S3

Budding Cells + + + + + + + +
True mycelium - - - - - - - -
Fragmenting - - - - - - - -
Pellicle - - - - - - - -
D-Glucose + + + + + + + +
D-Galactose + + + + - + - +
L-Sorbose - - - - - - - -
D-Ribose - - + - - - - -
D-Xylose - - +D - - - - -
L-Arabinose - - - - - - - -
Rhamnose - - - - - - - -
-Methylglucoside - - - - - - W +
Sucrose - - + + + + + +
Maltose - - - + + - - +
Trehalose - - - + - + - +
Cellobiose - - + - - - - -
Melibiose - - - + - - - -
Lactose - - + - - - - -
Raffinose - - - - + + + -
D-glucosamine - - - - - - - -
Acetyl-D-glucosamine - - + - - - - -
Soluble starch - - - + W - - +
Glycerol - - + - - - - -
Erythriol - - - - - - - -
Mannitol - - - - +D - - +D
Inositol - - - - - - - -
DL-Lactate - - - - + - - -
D-Gluconate - - +D - - - - -
D-Glucuronate - - - - - - - -
2-Keto-D-gluconate - - - - - - - -
Nitrate - - - - - - - -
Fermentation of + + + + + + + +
glucose
Ethylamine (N) - - - - - - - -
Vitamin requirement M M P M M 0B M M
Urease - - + - - - - -
Max. growth T (0C) 30 0C 30 0C 30 0C 40 0 C 40 0C 35 0C 37 0 C 37 0 C
Cycloheximide + + - - - +D - -
(100ppm)
+, positive; -, negative; W, weak response; D, delayed positive; V, variable; M, more or other vitamins required; P, pantothenate required; 0, no
vitamins required; B, biotin required.

Temperature Tolerance: 370C and feeble growth at 400C. As the temperature

The effect of temperature on growth of yeast isolates is
shown in Table-5. The strains S1, S2, S3, K1, K2 and
MTCC 170 have shown good growth upto 300C. C2 and
TA strains have shown good growth upto 370C, weak
growth at 400C and feeble growth at 420C. The strains
K2 and MTCC 170 showed similar growth pattern with
good growth upto 300C, moderate growth at 35 and
rises above 350C, the growth of the yeast strains
affected, complete inhibition of growth was observed
for S1, S2 and K1 strains from 37 to 450C. Temperature
tolerance of yeast was also studied by Patil and Patil
[22]. They have reported good growth of yeasts upto
370C and at higher temperatures growth was inhibited.
Similar results were obtained in our study. The inability

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of yeast isolates to grow at 450C in our study is in reported in literature [23].

agreement with the mesophilic character of yeasts well

Table 5. Effect of temperature on growth of yeast isolates on YEPD agar plates.

Temperature (0C)
Yeast Strains
25 28 30 35 37 40 42 45
S1 ++ ++ ++ - - - -
S2 ++ ++ ++ - - - -
S3 ++ ++ ++ - - -
TA ++ ++ ++ ++ ++ + -
C2 ++ ++ ++ ++ ++ + -
K1 ++ ++ ++ + - - - -
K2 ++ ++ ++ + + - -
MTCC 170 ++ ++ ++ + + - -
++, Good growth; +, Weak growth; , Feeble growth; -, No growth

Ethanol tolerance concentrations above 14% reduction in growth was

Ethanol generally inhibits growth and is toxic to cells.
As concentration of ethanol increases in media, a
reduction in growth is generally observed. This type of
behaviour is also shown by yeast strains in present
study. As there is a constant decrease in growth,
ethanol tolerance of a strain is taken at a concentration
of ethanol after which there is a sharp decrease in
growth. The results obtained for effect of ethanol on
growth are shown in Table-6, variation in ethanol
tolerance was observed among yeast strains.
In the present study S. cerevisiae C2 and TA strains
showed highest ethanol tolerance compare to other
strains. Both these strains were able to tolerate
maximum of 14% ethanol concentration in media. At
observed with multiple drops in intervals of optical
density values. The S. cerevisiae K2 strain was able to
tolerate maximum of 12% ethanol, and beyond this
concentration growth was decreased exponentially as
shown by drop in optical density values. S. rosinii S1
and S2 strains tolerated ethanol concentration upto
8%, followed by huge decrease in growth from 9%. R.
minuta S3 expressed similar ethanol tolerance and
tolerated maximum of 9% ethanol concentration,
followed by exponential decrease in growth observed.
Saccharomyces exiguus K1 strain tolerated slight higher
percentage of 10% ethanol. The reference strain S.
cerevisiae MTCC 170 was able to tolerate upto 12% of
ethanol in the media. At concentration above 12% a
sharp decrease in growth was observed.

Table 6. Ethanol tolerance of isolated yeast strains

Ethanol OD values at 600 nm after 48 hrs of incubation
Conc. (%) S1 S2 S3 TA C2 K1 K2 MTCC 170
1 1.50 1.75 1.78 1.80 1.70 1.79 1.60 1.62
2 1.42 1.50 1.60 1.76 1.68 1.65 1.56 1.60
3 1.34 1.38 1.50 1.72 1.62 1.58 1.54 1.56
4 1.30 1.30 1.40 1.70 1.60 1.45 1.50 1.52
5 1.26 1.00 1.00 1.65 1.58 1.30 1.45 1.50
6 0.90 0.94 0.96 1.62 1.52 1.18 1.42 1.46
7 0.70 0.75 0.82 1.60 1.50 0.98 1.40 1.40
8 0.40 0.50 0.70 1.50 1.48 0.80 1.30 1.35
9 0.25 0.30 0.45 1.48 1.45 0.72 1.26 1.30
10 0.15 0.22 0.30 1.46 1.40 0.40 1.00 1.10
11 0.10 0.15 0.22 1.44 1.40 0.25 0.90 1.00
12 0.02 0.09 0.10 1.36 1.32 0.16 0.81 0.90
13 0.0 0.04 0.04 1.30 1.28 0.10 0.70 0.75
14 0.0 0.0 0.0 1.24 1.20 0.04 0.50 0.50
15 0.0 0.0 0.0 1.15 1.10 0.0 0.30 0.40
16 0.0 0.0 0.0 1.00 1.00 0.0 0.20 0.35
17 0.0 0.0 0.0 0.6 0.50 0.0 0.10 0.20
18 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0

Ethanol is well known as an inhibitor of microbial Sugar Tolerance (Osmotolerance)

growth. It damages mitochondrial DNA in yeast cells
and causes inactivation of some enzymes, such as
hexokinase and dehydrogenase [24]. It was reported
that ethanol accumulation in fermenter inhibits specific
growth rate, specific ethanol production rate, cell
viability and substrate consumption [25]. Ethanol
tolerance of seven yeast strains isolated from fruits was
tested by Tikka et al., [26]. In their study they have
reported maximum of 12% ethanol tolerance by one of
the strain YDE. The results obtained in our study are
superior as two of the yeast strains S. cerevisiae TA and
C2 tolerated 14% ethanol.
The effect of sugar concentration on growth of yeast
isolates was studied in order to test the osmotolerance.
The sugar concentration was set up in the range of 10
to 35%.The results obtained are shown in Figure-1.
Among all strains, S. cerevisiae TA and C2 expressed
highest sugar tolerance of upto 20%, followed by
exponential decrease in growth which is shown by
huge drop in graph. The reference strain of S. cerevisiae
MTCC 170 tolerated a sugar concentration of 15%
followed by decline in growth. S. rosinii S1 and S2
strains and R. minuta S3 strain tolerated maximum of
10% sugar concentration which is comparatively less

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than S. cerevisiae TA and C2 strains. Similar to these yeast strains produce ethanol in range of 4.0 to 6.0 g/l
strains Saccharomyces exiguus K1 also tolerated 10% which is very less when compared with S. cerevisiae TA
sugar concentration. S. cerevisiae K2 strain expressed and C2 strains.
osmotolerance of 15% which is similar to
osmotolerance of reference strain MTCC 170. Keeping
in view data obtained when osmotolerance was
compared among yeast strains, it was observed that
potential yeast strains TA and C2 could tolerate a
maximum sugar concentration of 20%. The results
obtained in our study correlate with the data obtained
for sugar tolerance of wine yeasts by Osho A. [27] who
also reported maximum of 20% sugar tolerance for S.
cerevisiae BSOSU 0269.

Bioethanol production
In the present study after studying temperature
tolerance, osmotolerance and ethanol tolerance of
yeast strains, ethanol production ability was also
studied by batch fermentation in order to find out the
potential ethanol producers. The results obtained for
ethanol production are presented in Table-7. Among all
the strains, two strains S. cerevisiae TA and C2 were
found to be potential ethanol producers as they have
produced highest amount of ethanol (32.0 and 28.0 g/l
respectively). During the process of the fermentation,
ethanol production increased with increase in time
from 24 hrs to 72 hrs with highest production at 72 hrs
of fermentation, this pattern was observed for all the
strains studied. The reference strain S. cerevisiae MTCC
170 produced 20.0 g/l of ethanol after 72 hrs of
fermentation; S. cerevisiae K2 produced lesser amount
of ethanol (15.0g/l) than the reference strain. The other
Figure 1. Effect of high sugar concentration on growth of
yeast strains
The results obtained in the present study indicate that
S. cerevisiae TA and C2 strains are efficient ethanol
producing strains with higher ethanol production
ability. This is in agreement upon comparison with the
results obtained for batch fermentation of ethanol
production by Gupta et al., [28], who have reported
maximum of 12.5% ethanol by S. cerevisiae (SCP1). In
an another study maximum ethanol production of
8.33% by S. ellipsoideus 101 was reported by Patil and
Patil [22].

Table 7. Ethanol produced and Left over sugar by fermentation (g/l)

Yeast Time of fermentation Alcohol Production (g/l) Left Over Sugar
Strains (g/l)
S1 24h 2.5 50.0
48h 3.2 26.0
72h 4.0 15.0
S2 24h 2.8 47.5
48h 4.0 20.0
72h 5.2 12.5
S3 24h 3.5 52.5
48h 4.2 30.0
72h 5.5 17.5
TA 24h 11.7 51.0
48h 20.0 32.5
72h 32.0 10.0
C2 24h 9.0 55.0
48h 18.0 36.0
72h 28.0 20.0
K1 24h 4.0 45.0
48h 5.2 30.00
72h 6.0 13.0
K2 24h 7.0 12.5
48h 8.6 62.5
72h 15.0 22.5
MTCC 24h 7.5 60.0
170 48h 13.0 40.0
72h 20.0 17.5

The results of left over sugar are shown in Table-7, as
fermentation time increases decrease in left over sugar
concentration was observed which co-related with
simultaneous increase in ethanol production. The least
amount of left over sugar was recorded at 72 hrs of
fermentation for all yeast strains. Upon comparison of
data obtained for left over sugar, it was observed that
all yeast strains utilized more or less similar amounts of
sugar during the fermentation period (72 hrs) but not
all strains are efficient ethanol producers. Three out of
seven yeast strains including MTCC 170 are efficient
ethanol producers- S. cerevisiae TA and C2 These

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results once again prove that among all yeasts, S.
cerevisiae is more successful for ethanol production
when compared to other species [29]. This is due to the
fact that some species adopt different metabolic
pathways by having special genes or special enzymes
such as invertase genes and invertase enzymes
respectively for the conversion of sugars to ethanol or
other metabolites [30].
Results presented in Table-8 indicate that the growth
and fermentation kinetics with yeast strains S.
cerevisiae TA and C2 was faster than with other yeast
strains and with these strains higher ethanol
concentrations were achieved. The final ethanol
concentration (P) obtained for S. cerevisiae TA and C2
strains (32.0 and 28.0 g/l respectively) were much
higher than other four strains (P range from 4.0 to 15.0
g/l). These higher concentrations of ethanol were due
to higher conversion rate into ethanol (16.8 & 15.5 %)
by these yeast strains. Similarly, enhancement in
volumetric substrate uptake (QS= 2.638 & 2.50 g L-1 h-1)
and volumetric product productivity (Qp=0.44 & 0.38 g
L-1 h-1) was recorded with TA and C2 strains than other
strains including MTCC strain. The ethanol yield
(Yp/s=0.16 & 0.155 g g-1) obtained for S. cerevisiae TA
and C2 strains was found to be higher than that of other
yeast strains (Yp/s range from 0.047 to 0.068).

Table 8. Growth and Fermentation kinetics of yeast isolates in stationary fermentation

Yeast Strains
Paramaters
S1 S2 S3 TA C2 K1 K2 MTCC
170
Final ethanol (P, g L-1) 4.0 5.2 5.50 32.0 28.0 6.0 15.0 20.0
Final Biomass concentration (X, g L-1) 2.80 2.50 3.50 3.0 2.60 3.80 2.40 3.0
Cell yield (Yx/s, g/g) 2.37 2.06 3.07 1.14 1.04 3.16 1.35 1.63
Ethanol Yield (Yp/s g g-1) 0.047 0.059 0.066 0.160 0.155 0.068 0.110 0.150
Volumetric substrate uptake (QS, g L-1 h-1) 1.180 1.215 1.145 2.638 2.50 1.20 1.77 1.840
Volumetric product productivity (Qp, g L-1 h-1) 0.05 0.07 0.076 0.44 0.38 0.08 0.20 0.27
Conversion rate into ethanol (%) 4.70 5.90 6.60 16.80 15.50 6.80 11.0 15.0

CONCLUSION 5. Deepak S. (1994). Isolation and selection of thermotolerant

Based on the results obtained for identification studies
we can conclude that these seven isolates belong to
four species of yeasts: S1 and S2 belong to
Saccharomyces rosinii species, S3 belong to Rhodotorula
minuta species, TA, C2 and K2 belong to Saccharomyces
cerevisiae species and K1 belongs to Saccharomyces
exiguus species. Based on the data obtained for
characterization studies and fermentation kinetics we
can conclude that out of seven yeasts, two yeasts
Saccharomyces cerevisiae TA and C2 are high ethanol
tolerant, high osmotic tolerant and high bioethanol
yielding strains which can be exploited in future by
industries for bioethanol production.
Acknowledgements
The authors are thankful to Principal and Management
Committee, Mumtaz Degree and P.G College,
Hyderabad, India for providing facilities to carry out
the work.
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