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Ethanol production from cane-molasses by a thermotolerant strain of

Saccharomyces cerevisiae.

Conference Paper · August 2010

DOI: 10.13140/2.1.2580.5601

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 Ethanol production from cane-molasses by a thermotolerant
strain of Saccharomyces cerevisiae.

M. H. Vohra, R. V. Burase, S. P. Takle and S. V. Patil*

Vasantdada Sugar Institute, Manjari (Bk.), Tal. Haveli, Pune-412 307, India.
-----------------------------------------------------------------------------------------------------------

Abstract:
The aim of this work was to develop an economical process to produce ethanol
from cane-molasses at higher temperature using 14-liter bioreactor. Thermotolerant strain
of Saccharomyces cerevisiae (TSC-20073) isolated at VSI was used to ferment ethanol
from molasses under conditions of uncontrolled temperature and at controlled
temperature of 33°C. Maximum yield of 302.23 L/ton of molasses and fermentation
efficiency of 90.14 % were obtained during the experiment with molasses-2. GC analysis
of distillate at the end of fermentation showed a maximum fusel oil content of 560.22
PPM obtained under uncontrolled temperature condition using molasses-2.
------------------------------------------------------------------------------------------------------------
Keywords: - Saccharomyces cerevisiae, thermotolerant, bioreactor, productivity, fusel
oil.
* Technical Adviser & Head, Department of Alcohol Technology, VSI, Pune, India,
(sv.patil@vsisugar.org.in).

1 Introduction:
Alternative energy resources such as ethanol are becoming more important due to
depletion and negative environmental effect of fossil energy sources like coal and
petroleum. A worldwide interest in the utilization of bio-ethanol as an energy source has
stimulated studies on cost and efficiency of industrial bio-ethanol production processes.
Even though the alcoholic fermentation process is well known, intense research is being
carried out for obtaining efficient fermentative organism and optimal conditions for
fermentation (1).
Ethanol production by thermotolerant yeast has been extensively studied, because
of possible reduction in expensive cooling cost required to maintain temperature for
fermentation. Thermotolerant yeast capable of growth and fermentation at high

1
temperature is of much importance especially in tropical countries such as India, where
summer temperatures are quite high (2). Research is also being carried out on the usage
of thermotolerant yeast in simultaneous saccharification and fermentation of cellulose
where condition of high temperature is required (3, 4). Other advantages of processing at
high temperature include the possible use of continuous ethanol stripping as a method of
harvesting ethanol, significant reduction of contamination chances and reduction in the
volume of distillery cooling-wastewater effluent (2).
The demand for higher temperature fermentation began in the 1980s during which
many studies reported the screening of thermotolerant yeast strains with the capacity to
undertake ethanol fermentation under the conditions of simultaneous saccharification and
fermentation (SSF). Hacking et. al. (5) screened total 55 yeast strains for their ability to
ferment glucose to ethanol at elevated temperatures. They achieved yields of 50% of the
theoretical maximum with 28 strains at 37°C but only 12 strains at 40°C. Six strains
could grow at 45°C but gave a poor yield of ethanol at this temperature. Thermotolerant
strains of Saccharomyces, Kluyveromyces, and Fabospora genera have also been
identified that can grow at temperatures above 40°C and ferment sugars at 40°C, 43°C,
and 46°C, respectively (6). Another study has described 11 Saccharomyces and
Kluyveromyces strains, identified from among 65 strains of seven yeast genera that have
good growth ability and can ferment ethanol at 40°C (7).
Among the known yeast species used in fermentation processes, Kluyveromyces
marxianus is thought to have the best performance in terms of growth and fermentation at
high temperatures. It has also been experimentally documented that many thermotolerant
strains of K. marxianus grow well at temperatures as high as 45°C to 52°C and can
efficiently ferment ethanol at temperatures of between 38°C and 45°C (8, 9, 10, 11, 12).
Thermotolerant strains of Saccharomyces cerevisiae have also been screened that can
tolerate temperature above 44°C but growth and ethanol production levels in these cases
were found to decrease at 44°C (13). Torija et. al. (14) reported the influence of
fermentation temperature on growth, viability and fermentation of S. cerevisiae from
15°C to 35°C. It was observed that at 35°C a high amount of yeast died, indicating that at
high temperature decreased yeast viability leads to lower growth and lower fermentation
yield.

2
 We have isolated and screened number of thermotolerant strains of S .cerevisiae
in our laboratory (15). Thermotolerant strain of S. cerevisiae (TSC-20073) was selected
for further studies. The main objective of this work was to develop and optimize an
economical bioprocess to produce ethanol from cane molasses by the selected strain at
10-liter scale. Fermentation kinetic was studied under various conditions of controlled
and uncontrolled temperatures. Yields and efficiencies have been determined.

2.0 Materials and Methods:

2.1 Characterization of molasses:
Molasses of differing composition from two different factories viz. Shree
Someshwar S. S. K. Ltd., Baramati, MS, India (M-1) and T. K. Kore Warana S.S.K. Ltd.,
Kolhapur, MS, India (M-2) were used for experimental purpose. Both the molasses were
stored at room temperature and were diluted with water prior to fermentation
experiments.
The total reducing (TRS) and unfermentable sugars (UFS) of molasses were
determined by Lane and Eynon method (16). The composition of sugars was determined
by HPLC (Waters 515) using Sugar-Pak I column (6.5 mm X 300 mm) maintained at
90°C in an oven. Deionized water was used as a mobile phase at 0.5 ml/min.
The percentage (w/v) of soluble solids (Brix°) was determined by brix
hydrometer. The specific gravity of molasses was determined by specific gravity
hydrometer. Moisture content was determined by gravimetric analysis after drying at
105°C to constant weight. Ash content was quantified by gravimetric analysis by burning
sample at 550°C for 2 h. Suspended solids was determined by gravimetric analysis after
filtering the diluted molasses and drying the filter paper at 105°C to constant weight. The
sludge content was determined by centrifuging 1:1 diluted and boiled molasses at 3000
rpm for 20 mins. The supernatant was discarded and sludge content in the centrifugation
tube was measured. The calcium content was estimated by EDTA method (17) while the
total organic volatile acidity was determined by the method described in Standard
methods for the examination of water and wastewater (18).

3
2.2 Yeast source:
Thermotolerant Saccharomyces cerevisiae (TSC-20073) strain isolated in our
laboratory from distillery environment was maintained on MGYP agar slant having
composition: malt extract 3 g/L; yeast extract 3 g/L; glucose 20 g/L; peptone 5 g/L; agar
20 g/L. In all cases, slant culture was incubated at 32.5oC for 24 hours and then
maintained at 4oC. Sub culturing was done after every month.

2.3 Inoculum development:

The inoculum for fermentation experiments was grown in 12 Brix molasses
medium containing 0.01% Diammonium phosphate and 0.01% Urea and having pH 4.5
adjusted with 10 % Sulphuric acid. The 500 ml and 2000 ml capacity Erlenmeyer flasks
containing 100 ml and 800 ml of above medium respectively were sterilized in an
autoclave at 1.1 bar pressure for 15 minutes. Two 100 ml of sterilized molasses medium
flasks were inoculated with loopful of fresh yeast slant culture and were incubated on
rotary incubator shaker for 16 h at 30C temperature and 180 rpm. These flasks were
transferred aseptically to a 800 ml of above mentioned sterilized molasses medium to get
a final volume of 1000 ml. The medium was incubated on rotary shaker for 24 h at 30C
temperature and 180 rpm and was used as final inoculum (seed culture). Inoculum was
analyzed for viability of cells.

2.4 Ethanol production medium:

Both the molasses were diluted with raw water so as to obtain final concentration
of 17.5 g % TRS. The molasses were then supplemented with 0.01 % DAP and 0.01 %
urea, pH adjusted to 4.6 and used as ethanol production (EP) medium (15). This EP
medium was used in unsterlized condition.

2.5 Fermentation conditions:

Fermentation experiments at 10-liter working-volume were carried in a 14-liter
stainless steel bioreactor (Chemap, Switzerland) equipped with agitation, temperature and
pH controller/indicators and fitted with a reflux cooler in the exhaust line to minimize
evaporation. The vessel was filled with 9 liter of EP medium. This unsterlized EP

4
medium was inoculated with 1 liter of seed culture to give a final volume of 10 liter. The
fermentation was carried out using molasses either at uncontrolled temperature starting
with room temperature of 33C (temperature allowed to increase as fermentation
proceeded) or at a controlled temperature of 33C. The temperature was maintained by
pumping water of 21C through the water jacket of the fermenter. The pH was set at 4.6
and allowed to change as fermentation proceeded without controlling it. Agitation of 70
rpm was provided for all the experiments. Sampling was done at regular intervals till the
fermentation was complete.

2.6 Analytical method:

Biomass concentration (cells/ml) was determined by using haemocytometer (19).
The viability of yeast cell was determined by methylene blue staining. The total reducing
(TRS) sugars of fermentation medium was determined by Lane and Eynon method (16).
Ethanol concentration was determined from distillate using pycnometer (20). Ethanol as
well other by-products from distillate were analyzed by injecting 1.5 l of sample in Gas
Chromatograph (Hewlett Packard 6890 plus, column; capillary column Stabilwax-Da,
injection temperature; 240C, carrier gas; nitrogen, detector; flame ionization with 260C
temperature).

2.7 Calculation of kinetic parameters:

The maximum theoretical efficiency was calculated according to the
stoichiometric relation represented by Eq. (1), i.e.100 g hexose produces 51.1 g (64.4 ml)
of ethanol and 48.9 g of CO2. Fermentation efficiency from initial total fermentable sugar
is calculated according to Eq. (2)
C6H12O22  2CH3CH2OH +2CO2 (1)

Fermentation efficiency, % = Pf, % (v/v) x 100 (2)

[So, g %] x 0.644
Where, Pf = final ethanol concentration, % (v/v); So= initial total fermentable sugar
concentration, g %.

5
 Fermentation yield was also determined as ethanol produced per metric ton of
molasses.
3.0 Results and Discussion:
3.1 Characterization of molasses:
The composition of both the cane molasses samples used is shown in Table-1. It
can be seen that both the molasses has high amount of volatile fatty acids. Sludge content
of molasses-1 was also substantially high. Fermentable sugar in molasses-2 was
substantially high with F/N ratio of 1.92.

Table-1: Molasses analysis report

Sr.No. Parameter M-1 M-2

o
1 Brix 93.5 88.3
2 Specific gravity 1.320 1.300
3 Moisture, % 27.35 20.88
4 Total suspended solids, % 14.54 8.62
5 Total dissolved solids, % 58.11 70.50
6 pH 4.39 4.85
7 Carbonated ash, % 11.555 8.675
8 Sulphated ash, % 14.155 10.150
9 Total reducing sugar (T.R.S.), % by weight 51.78 55.14
10 Total unfermentable sugar (U.F.S), % by weight 4.38 3.10
11 Total fermentable sugar, % by weight 47.40 52.04
12 Fermentable/Non fermentable ratio (F/N) 1.87 1.92
13 Sludge, %
pH adjustment, 1:1dilution, Heat to 90 C 3.0 1.5
Centrifuge at 3000 rpm, 20 minute.
pH adjustment, 1:1 dilution, Heat to 90 C, 1 23 12
hour settling
14 Calcium, grams/100 Bx. 2.43 2.22
15 Total Organic Volatile Acids, mg/lit 9342.85 5314.28
16 Glucosea, % 4.177 7.915
17 Fructosea, % 8.650 9.232
18 Sucrosea, % 33.150 37.455
Total G+F+S, % 45.977 54.602
19 Raffinosea, % 0.733 1.089
20 Xylosea, % 0.035 ND
a
By HPLC, ND- not detected.

6
3.2 Ethanol production using M-1 at uncontrolled temperature (E-1).

The fermentation experiment was carried out at uncontrolled temperature starting

with 33C. Fig. 1 illustrates the temperature and pH profiles during the fermentation
process. In our previous studies it was established that the thermotolerant yeast strain
(TSC-20073) could efficiently ferment molasses up to 40C. In this experiment, the
temperature of fermentation was allowed to increase without any control. The effect of
increasing temperature on yeast cell growth, sugar consumption rate and ethanol
production rate was also checked.

Fig. 1 Temperature and pH profile at uncontrolled temperature using

M-1
Temperature,°C pH

40 5
39 4.9
4.8
Temperatue, °C

38
4.7
37 4.6

pH
36 4.5
35 4.4
4.3
34
4.2
33 4.1
32 4
-1 0 1 2 3 4 5 6 7 9 10 12 16 18 21 22 25
Time, h

Fig. 2 Sugar consumption and ethanol production at uncontrolled

temperature using M-1
Total reducing sugar, % Ethanol concentation, % (v/v)

20 10
Ethanol concentration,

18 9
Total reducing sugar,

16 8
14 7
% (v/v)

12 6
g%

10 5
8 4
6 3
4 2
2 1
0 0
0 5 10 16 22 25
7
Time, h
 From Fig. 1, it is seen that the temperature of fermentation increased up to
maximum of 37.2C at around 12 hours of fermentation. After that, the temperature
slowly decreased and reached to 36C. The pH of the fermentation medium decreased to
4.33 after 16 h of fermentation and then remained constant.
Sugar consumption and ethanol production during E-1 is shown in Fig. 2. The
sugar was almost completely consumed under uncontrolled fermentation temperature.
The fermentation was completed in 25 h with ethanol volumetric yield of 9.40 % (v/v).

3.3 Ethanol production using M-1 at controlled temperature of 33C (E-2).

This experiment was performed to check the effect of controlled temperature of

33C on the ethanol production. The pH profile during the experiment was almost similar
to the previous experiment.
Sugar consumption and ethanol production during E-2 is shown in Fig. 3. The
fermentation was completed in 30 h, as compared to 25 h at uncontrolled temperature
experiment with ethanol volumetric yield of 9.54 % (v/v). This indicates that the rate of
fermentation by TSC-20073 increases as the fermentation temperature increases.

Fig. 3 Sugar consumption and ethanol production at controlled

temperature of 33°C
Total reducing sugar, % Ethanol concentation, % (v/v)

20 10
18
concentration, %

16 8
Total reducing

14
sugar, g%

Ethanol

12 6
(v/v)

10
8 4
6
4 2
2
0 0
0 5 10 16 22 26 28 30
Time, h

8
3.4 Ethanol production using M-2 at uncontrolled temperature (E-3).

The fermentation experiment was carried out at uncontrolled temperature starting

at 33C. Fig. 4 illustrates the temperature and pH profiles during the fermentation
process.

Fig. 4 Temperature and pH profile at uncontrolled temperature using

M-2
Temperature,°C pH

41 5
40 4.9
Temperatue, °C

39 4.8
38 4.7
37 4.6

pH
4.5
36 4.4
35 4.3
34 4.2
33 4.1
32 4
-1 0 1 2 3 4 5 6 7 10 12 13 16 17 19 21 24 27 30
Time, h

From fig. 4 it can be seen that the ambient temperature before the start of the
experiment was 38.4C as compared to 33C during E-1. In all experiments the
temperature of fermenter was brought down to 33C. It can be seen that because of higher
ambient temperature and heat generated during alcoholic fermentation, the temperature of
the fermenter quickly increased and reached to a maximum of 40C after 12h of
fermentation (E-3). The pH profile of E-3 experiment is also different when compared
with molasses 1 (E-1). The pH of E-3 reached to 4.24 which is lower than that reached
during E-1, which was 4.33. This might be due to slightly longer fermentation time
required during E-3.
Sugar consumption and ethanol production during E-3 is shown in Fig. 5. It can
be seen that ethanol volumetric yield reached to 9.59 % (v/v) after 30 h of fermentation,
slower than E-1. This might be due to the fermentation temperature, which reached to
40C. At this temperature, effect of ethanol stress on yeast cell might be increasing.

9
However, the volumetric yield is higher when compared with reported yields using
thermotolerant strain of mutant S. cerevisiae and K. marxianus at 40C (21).

Fig. 5 Sugar consumption and ethanol production at uncontrolled

temperature using M-2
Total reducing sugar, % Ethanol concentation, % (v/v)

20 10
18
Total reducing

concentration,
16 8
14
sugar, %

Ethanol

% (v/v)
12 6
10
8 4
6
4 2
2
0 0
0 5 10 16 22 28 30
Time, h

3.5 Ethanol production using M-2 at controlled temperature of 33C (E-4).

This experiment was performed to check the effect of controlled temperature of

33C on the ethanol production. The pH profile was similar to previous experiment (E-3).
The pH at the end of fermentation reached to 4.28. Sugar consumption and ethanol
production during batch fermentation using molasses 2 at controlled temperature of 33C
is shown in Fig. 6.

Fig. 6 Sugar consumption and ethanol production at controlled

temperature of 33°C using M-2
Total reducing sugar, % Ethanol concentation, % (v/v)
20 10
concentration, % (v/v)

18
Total reducing

16 8
sugar, g%

14
Ethanol

12 6
10
8 4
6
4 2
2
0 0
0 5 10 16 22 28 32
Time, h

10
 The sugar was almost completely consumed during the fermentation experiment
at 33C. The fermentation was completed in 32 h, which is slightly higher than at
uncontrolled experiment (E-3) with ethanol volumetric yield of 9.40 % (v/v).
Comparison of ethanol production parameters using molasses-1 and molasses-2
during batch fermentation at uncontrolled temperatures and at 33C is given in Table-2.
GC analysis of distillate obtained at end of fermentation is given in Table-3. It can be
seen that maximum yield of 302.23 L/ton of molasses was achieved under uncontrolled
temperature condition using M-2. With uncontrolled temperature conditions,
fermentation efficiencies obtained with both M-1 and M-2 molasses were above 90 %,
which indicates that the thermotolerant strain (TSC-20073) can be used easily for
commercial scale fermentations.

Table-2: Comparative ethanol production parameters using M-1 and M-2 during
batch fermentations at uncontrolled temperatures and at 33C

Experiment Fermentation Maximum Total Final Yield, Efficiency,

time required, temp fermentable Ethanol L/ton %
h reached, C sugar, g % Conc.,
% v/v
E-1 25 37.2 15.96 9.40 278.18 91.45
E-2 30 33.0 15.96 9.54 282.32 92.81
E-3 30 40.0 16.52 9.59 302.23 90.14
E-4 32 33.0 16.52 9.40 296.24 88.35

The GC profiles of the distillate for all the experiment are qualitatively almost the
same. Quantitatively Fusel oil production is higher in uncontrolled temperature
experiments (E-1 and E-3) than experiments carried at 33C (E-2 and E-4).

11
 Table-3: GC analysis of distillate obtained at end of fermentation

Name Amount in mg/l

E-1 E-2 E-3 E-4
Acetaldehyde 115.4 204.94 87.785 ND
Acetone 4.724 3.715 7.744 ND
Ethyl acetate 143.71 132.03 126.65 22.602
Methanol 6.308 5.900 8.6275 ND
2-Propanol ND ND ND ND
Diacetyl 56.017 50.170 26.871 23.343
2-Butanol ND ND ND ND
1-Propanol 90.821 63.038 123.62 29.092
2, 3 Pentanedione 4.474 6.003 ND ND
N-Butyl acetate ND ND ND ND
Iso-Butanol 40.346 27.639 78.450 11.826
1-Butanol 3.531 2.072 8.848 ND
Iso-amyl alcohol 187.31 124.61 349.31 60.943
1-Pentanol ND ND ND ND
Acetic acid ND ND ND 107.97
Furfural 1.270 1.673 1.1001 ND
Fusel oil 326.30 223.36 560.22 101.86
ND- not detected

4. Conclusion:

10-liter scale experiments using thermotolerant strains under condition of

uncontrolled temperature are seen to be promising. In distilleries where cost saving is the
main agenda, performing fermentations under uncontrolled temperature conditions using
TSC-20073 would be of great use. Our thermotolerant yeast strain produced ethanol with
a maximum yield and efficiency of 302.23 L/ton and 90.14 %, respectively. Maximum
temperature reached during the experiment depends on the ambient temperature.
Maximum temperature of 40C was reached under uncontrolled fermentation conditions
during which yeast viability, ethanol production yield and efficiency did not decrease.
Thus our yeast has the capacity to perform fermentation at higher temperature, which can
be very important from cost economic point of view in distilleries.
Existing distillery units can expect a saving of around Rs. 10 to 11 Lakhs per
annum as given below. For new distilleries, apart from saving of Rs. 10 to 11 Lakhs per
annum, savings in terms of capital investment of Rs. 30 Lakhs can be also expected.

12
Cost Economics of using TSC-20073 for 30 KLPD distillery capacity:
Existing distillery:
Savings in Electricity cost @ Rs 4.50/unit x 720 units/day: Rs. 3,240/- per day
Savings in maintenance and operation cost: Rs. 120/- per day
Saving in water evaporation loss, 130 m3 per day x Rs. 10/ m3 Rs. 1300/- per day
New distillery:
Savings in Cooling tower cost: Rs. 8,00,000/-
Savings in PHE cost: Rs. 18,00,000/-
Savings in Interconnecting pipeline cost: Rs. 4,00,000/-
Savings in Electricity cost @ Rs 4.50/unit x 720 units/day: Rs. 3,240/- per day
Savings in maintenance and operation cost: Rs. 120/- per day
Saving in water evaporation loss, 130 m3 per day x Rs. 10/ m3 Rs. 1300/- per day

5. Acknowledgements:

The authors sincerely acknowledge the support and encouragement provided by

Mr. Shivajirao Deshmukh, Director General, VSI for the research work related to
thermotolerant yeast.

5. References:

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