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Studies on optimization conditions for alcoholic
fermentation process of delta beet molasses
A. A. Zohri a, A. M. Ramadan b, M. M. El-Tabakh c and K. El-Tantawy c
a)
b)
Assiut University ,Faculty of Science, Egypt
Kafr El-Sheikh University, Faculty of Science, Egypt
c)
Delta Sugar Company, Egypt
Abstract:
A mixture sample of beet molasses from production line I of DELTA sugar
factory at period from 9/2 to 12/2 during season 2010 was used as a good
fermentation medium in this study. Sugar levels of molasses were adjusted to
different concentrations (5, 10, 15, 20, 25 and 30%) using distilled water. The
results appeared that the highest level of ethanol production was 9.2% (equal
90% of theoretical values). This level was recorded when used molasses with
20% sugar and grown for 72 hours. To know the optimum temperature for
ethanol fermentation, the fermentation process was kept at 25, 30, 35 and 40˚C.
Ethanol yield reached to maximum and turned out to be 9.2% after 48 hours at
30˚C. Also, effect of pH on ethanol production level was examined and the
results appeared that the maximum ethanol level was obtained at pH 5.
Introduction:
During industrial scale of ethanol fermentation yeast- encounters a
multitude of stress factors that impose constrains on yeast growth as well as
fermentative metabolism. These stresses include high sugar concentration,
elevated temperature, high ethanol concentration and low external pH. So, this
investigation was designed to study the effect of each of sugar concentration,
temperature and pH on the concentration of ethanol production using DELTA
beet molasses.
Use of concentrated sugar substrate is one of the ways to obtain high
ethanol yield during fermentation. However, Jones et al. (1994) found that the
high sugar concentrations are inhibitory to fermentation due to osmotic stress.
Borzani et al. (1993) studied fermentation with various initial concentrations of
sugar and demonstrated the logarithmic relationship between time of
fermentation and initial concentrations of sugar. Bertolini et al. (1991) isolated
yeast strains had the ability to ferment up to 30% sucrose efficiently from sample
collected from Brazilian alcohol factories. They recorded that the efficiency of
selected strains varied from 89% to 92% depending upon the utilization of total
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sugar available in the medium with maximum amount of 19.7% (v/v) ethanol
accumulated. A repeated batch fermentation system was used to produce ethanol
using an osmotolerant S.cerevisiae (US3) immobilized on calcium alginate (Sree
et al., 2000). Their fermentation was carried out with initial concentration of 150,
200, 250 g glucose per liter at 30ºC and the maxima amounts of ethanol
produced were 72.5, 93 and 83 g ethanol per liter at 30ºC after 48h.
The fermentation process is always accompanied with evolution of heat
that raises the temperature of the fermentor. As a result it becomes necessary to
cool the large fermentors in the distilleries. This necessity often becomes a major
operation and a cost factor in the production of ethanol. Temperature exerts a
profound effect on growth, metabolism and survival of the fermenting organism.
Fermentation in industries is usually carried out at ambient temperature of 2535ºC but temperature exceeds 40ºC during fermentation especially in northern
regions which decreases the cell viability and productivity. Maintenance of high
cell viability is a major characteristic of fermentation to get high ethanol yield.
Fermentation at 35-40˚C or above has advantages such as ethanol recovery
and significant savings on operational costs of refrigeration control in distilleries
for alcohol production. Therefore many studies have been carried out for
development of yeast to ferment at high temperature of up to 40-45˚C. Laluce et
al. (1993) studied the effects of temperature on fermentation capacity of three
yeast strains 19G, 78I and baker’s yeast in complete medium and sugarcane juice
broth containing 15% total sugar. They reported that a complete conversion of
total sugar to ethanol was observed after 12 hrs of fermentation at 39-40˚C. They
also observed that above 40˚C, a strong inhibitory effect on ethanol production
was occurred.
Singh et al. (1998) studied the ethanol production at elevated temperatures.
They isolated a number of strains of Kluyveromyces marxiaus var. marxianses
capable of growth at high temperatures coupled with production of high alcohol
concentrations by fermentation of glucose and molasses.
Morimura et al. (1997) made an attempt to improve the salt tolerance of
the thermotolerant flocculating yeast Saccharomyces cerevisiae strain KF-7 by
maintaining a high concentration of KCl in the molasses medium. Among
selected strains, K211 had the highest cell viability and ethanol productivity in a
molasses medium containing 25% (w/v) total sugar at 35˚C.
Yadav et al. (1997) found an increase in alcohol concentration
productivity as well as efficiency with an increase in pH from 4.0-5.0 and found
that the optimum pH range for S. cerevisiae to be between pH 4.5 - 5.0. Early,
Prescott and Dunn (1959) reported that the yield of 90% ethanol or more of the
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theoretical values were obtained under the optimum fermentation condition. They
also reported that the efficiency of ethanol fermentation depends on the pH and
they suggested that a pH of 4 to 4.5 favour the yeast growth, but is sufficiently
low to inhibit the development of many type of bacteria.
Russell (2003) recorded that yeast prefers an acid pH and its optimum pH
is 5.0 – 5.2. Narendranath and Power (2005) found that the optimum pH for
yeast growth and ethanol production by S. cerevisiae was pH 4.9. Limtong et al.
(2007) examined the ethanol production from sugar cane juice with 22% sugar
and found that the highest ethanol concentration was obtained when the
fermentation carried out at pH 5.0.
Material and methods
Molasses Samplers
A mixture sample of beet molasses from production line I of DELTA sugar
factory at period from 9/2 to 12/2 during season 2010 was collected and used as a
good fermentation medium in this study.
Yeast Strain
Yeast strain was Saccharomyces cerevisiae AU 71 (thermotolerant and
high ethanol producing strain) which kindly obtained from Industrial
Microbiological Laboratory, Botany Department, Faculty of Science, Assiut
University.
Optimization of fermentation process
The collected mixture sample of beet molasses was supplemented with
0.1% KH2PO4 & 0.2% (NH4)2SO4 and used in the following experiments for
optimized the ethanol production conditions from beet molasses.
a) Effect of sugar concentration
To study the effect of sugar concentration on ethanol production by
S.cerevisiae, the production media was prepared by diluting selected molasses to
sugar concentration of 5, 10, 15, 20, 25, 30 percent with distilled water. The
diluted samples of beet molasses were heated at 95˚C for 20 min, then cooled and
filtered through ordinary filter paper to remove suspended particles. The prepared
inoculum of selected yeast strain was added at the rate of 10 percent to 100 ml of
the medium in 200 ml sterilized bottles. Prepared molasses media were adjusted
to pH 5.0. Fermentation carried out at 120 rpm and 30˚C for different incubation
periods up to 72 hours under anaerobic condition. Samples were withdrawn after
every 12 hours interval and determined the dry yeast weight as will as ethanol
content in the fermentation medium. The percentage of ethanol was calculated.
The initial sugar concentration that was efficiently utilized by the yeast for
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ethanol production was selected for further experiments.
b) Effect of temperature
To optimize the fermentation temperature, fermentation was carried out at
25, 30, 35 and 40ºC. Molasses diluted to 20% sugars. Prepared molasses were
heated at 95˚C for 20 min, thin cooled and filtrated. The pH was adjusted to 5.0.
Fermentation was carried out in 200ml sterilized bottles which had 100 ml of
prepared molasses medium and inoculated with prepared inoculum of selected
yeast at rate of 10%. Cultures were incubated at 120 rpm for 48 hours. The
periodic samples were analyzed for dry yeast weight and ethanol content.
c) Effect of pH
Different levels of pH (5.0, 6.0, 7.0 and 8.0) were tested for fermentation
using molasses with 20% sugar concentration. Prepared molasses were heated at
95˚C for 20 min, then cooled and filtered before adjusted the pH. Fermentation
was carried out in 200ml sterilized bottles (had 100 ml of prepared molasses
medium and inoculated with prepared inoculum of selected yeast at rate of 10%)
at 30˚C and 120 rpm for 48 hours. Periodic samples (at each 12 hours) were
analyzed for dry yeast weight and ethanol content.
Analytical concepts
a) Physicochemical analysis: Each of brix%, specific gravity, viscosity, colour
brix %, pH, total sugar%, non-fermentable sugar%, fermentable sugar% and
sulfated ash% were analyzed using analytical methods of International
Commission for Uniform Methods of Sugar Analysis (ICUMSA) at Delta
sugar factory, quality control department as routine and classical quality
control analysis.
b) Detection of Ethanol: Ethanol contents were estimated by dichromate
method described by Zohri and Eman Mostafa (2000).
c) Detection of yeast cells viability: Direct viable cell count was carried out
using a hemacytometer after staining with 0.1% methylene blue (Borzani and
Vario, 1958). Purity of yeast cells was examined by light microscope.
d) Determination of yeast growth: Dry biomass was measured by filtered the
yeast biomass using known weight filter paper, washed three times with
distilled water and dried for 24 hours at 85˚C. Dry yeast biomass was
calculated from re-weight the dried filter paper plus biomass.
e) Detection of pH value: The pH measures were performed directly using a pH
meter (Microprocessor pH – mV meter pH 526).
f) Determination of sugar levels: sugars were determined by the method
described by the European Community (2000) as official methods for
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detection of sugar in wines.
Results and Discussion
Average of Physicochemical analysis of Delta beet molasses sample:
A mixture sample of beet molasses from production line I of DELTA sugar
factory at period from 9/2 to 12/2 during season 2010 was used as a good
fermentation medium in this study. Physicochemical analysis of this sample was
achieved and the results recorded in Table (1). The result appeared the high level
of fermentable sugar and their suitability for ethanol fermentation.
9/2 to
12/2
80.0
1.4142
1265.0
Brix
31320
pH
sugar%
9.5
53.7
0.90
52.80
sulfated ash%
(cp)
Total
sugar %
Brix% gravity
%
Fermentable
line I
Color
sugar %
specific Viscosity
Nonfermentable
Table (1): Average of Physicochemical analysis of Delta beet molasses collected from
the Line (I) at period from 9/2 to 12/2 during campaign 2010.
13.03
Optimization studies on fermentation of molasses:
a) Effect of sugar concentration
Sugar levels of molasses were adjusted to different concentrations (5, 10,
15, 20, 25 and 30%) using distilled water. Fermentation process was achieved in
200 ml bottles at 30˚C for 72 hours and incubated at 120 rpm. The results
obtained were recorded in (Table, 2) and illustrated in (Figure,1). The results
appeared that the highest level of dry yeast weight was 3.09 g/l and recorded
when used molasses with 20% sugar and grown for 72 hours (Table, 2). The
highest level of ethanol production was 9.2% (equal 90% of theoretical values)
and recorded after 48 hours when used molasses with 20% sugar (Table, 2 &
Figure, 1).
This result is harmony with those recorded by Maysa Ali (2010). She
examined the effect of sugar concentration (12.5, 15, 17.5, 20, 22.5, 25, 30 and
35%) on ethanol production by five yeast strains and reported that the maximum
ethanol level by Kluyveromyces marxianus GU133329 was 9.55% (equal 93.63%
of the theoretical value) at 20% sugar concentration. Zohri and Eman Mostafa
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(2000) studied the effect of different sugar concentrations (13.5, 18 and 22.5%) in
the date juice on the fermentation efficiency of two yeast strains and found that
the date juice with 18% sugars was the more suitable and economic
concentration. Gough et al. (1998) found that a sugar concentration more than
23% in molasses had a harmful effect on ethanol production.
As general, increasing sugar concentration (at 25 & 30%) lead to increase
the viscosity in fermentation medium and this had an inhibitory effect on yeast
growth and their capability to ethanol production. Similar results were recorded
previously in the fermentation medium with high gravity worts (Pratt- Marshall
et al., 2002, 2003). They observed that fermentation of high gravity worts have a
negative effect upon the yeast performance due to the elevated osmotic pressure.
Reddy and Reddy (2006) reported that the increasing in sugar concentration will
be decrease in sugar utilization, which results in reduction of the total ethanol
production. This reduction could be due to several reasons including the
production of other compounds like glycerol or acetic acid. Also, the intracellular ethanol (which may be increased by increasing ethanol production at high
sugar concentration) exerts high toxicity on yeast and the nutrient may be
deficiency (Sols et al., 1971).
Table (2): Effects of sugar concentrations on ethanol production from beet molasses
using S.cerevisiae AU71.
Dry yeast weight (g/l)
Ethanol Level %
Sugar concentration
Time
(hrs)
5%
10% 15% 20% 25% 30%
5%
0
0.08
0.14
0.24
0.39
0.45
0.44
0.0
0.0
0.0
0.0
0.0
0.0
12
0.75
0.70
0.72
0.66
0.55
0.73
2.0
3.2
3.6
4.4
3.5
3.6
24
1.22
1.10
1.08
1.54
1.34
1.18
2.3
3.8
4.2
7.8
6.9
7.0
36
2.2
1.89
1.94
2.14
1.55
1.45
2.5
4.5
5.8
8.3
8.1
8.5
48
2.54
2.44
2.64
2.89
1.79
1.86
2.5
4.9
7.5
9.2
8.6
8.5
72
2.86
2.75
2.76
3.09
1.98
1.98
2.5
4.9
7.5
9.2
8.6
8.5
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4
10
9
3.5
5%DY
8
10%DY
3
25%DY
30%DY
5%E.L
10%E.L
15%E.L
20%E.L
7
2.5
6
2
5
4
1.5
25%E.L
30%E.L
Ethanol level %
20%DY
Dry yeast weight(g/l)
15%DY
3
1
2
0.5
1
0
0
0
12
24
36
48
72
Time(hours)
Figure (1): Effect of sugar concentrations in molasses medium on dry yeast weight and
ethanol production by S.cerevisiae AU 71 grown at 30˚C for 72 hours on
120 rpm.
b) Effect of temperature
Temperature is one of the major constraints that determine the ethanol
production. To know the optimum temperature for ethanol fermentation, the
fermentation process was kept at 25, 30, 35 and 40˚C. The molasses were diluted
up to 20% sugars and fermentation was carried out in 200ml bottles, which
incubated at 120 rpm for 48 hours. Two parameters were simultaneously studied,
the growth of S.cerevisiae AU 71 and the ethanol yield. Samples were withdrawn
every 12 hours and the fermentation was carried out for 48 hours.
As shown in Table (3) and Figure (2) at 30˚C, ethanol yield reached to
maximum and turned out to be 9.2% after 48 hours. However increasing the
temperature beyond 30˚C, the growth as well as concentration of alcohol
decreased. This decrease was pronounced at 40˚C. Similar results were recorded
by Maysa Ali (2010). She reported that the highest ethanol levels by two
Saccharomyces cerevisiae strains were recorded at 30˚C. Reddy and Reddy
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(2006) recommended that the fermentation temperature for ethanol production up
to 30˚C should considered.
Table (3): Effects of temperatures on ethanol production from beet molasses using
S.cerevisiae AU 71.
Dry yeast weight (g/l)
Time
(hrs)
Alcohol (in %)
Temperature
25°C
30°C
35°C
40°C
25°C
30°C
35°C
40°C
0
0.23
0.25
0.24
0.23
0
0
0
0
12
0.48
0.74
0.56
0.48
1.1
4.4
2.9
1.1
24
0.98
1.45
1.23
0.75
2.9
7.8
5.1
2.1
36
1.32
1.89
1.56
0.98
5.3
8.3
6.9
2.5
48
1.86
2.83
1.9
1.2
6.9
9.2
8.1
3
25˚C (dry weight)"
3
30˚C (dry weight)"
14
35˚C (dry weight)"
40˚C (dry weight)"
12
2.5
25˚C(alcohol level %)
30˚C (alcohol level%)
10
2
40˚C (alcohol level%)
Dry yeast weight (g/l)
8
1.5
6
Alcohol level %
35˚C (alcohol level%)
1
4
0.5
2
0
0
0
12
24
36
48
Time(hours)
Figure (2): Effect of incubation temperature on dry weight and ethanol production by
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S.cerevisiae AU 71 grown on beet molasses with 20% sugar concentration
for 48 hours on 120 rpm in molasses medium.
c) Effect of pH
Initial sugar concentration in molasses medium of 20% and optimum
temperature of 30˚C was selected for further studies and subjected to pH
treatments 5, 6, 7 and 8. The results are shown in Table (4) and illustrated in
Figure (3). At pH 6, 7 & 8, fermentation took place but it gave low ethanol
content. Best results were obtained at pH 5 where maximum ethanol production
was noticed. Yadav et al. (1997) found an increase in alcohol concentration
productivity as well as efficiency with an increase in pH from 4.0-5.0 and found
that the optimum pH range for S. cerevisiae to be between pH 4.5 - 5.0.
Early, Prescott and Dunn (1959) reported that the yield of 90% ethanol or
more of the theoretical values were obtained under the optimum fermentation
condition. They also reported that the efficiency of ethanol fermentation depends
on the pH or mash and they suggested that a pH of 4 to 4.5 favour the yeast
growth, but is sufficiently low to inhibit the development of many type of
bacteria.
Russell (2003) recorded that yeast prefers an acid pH and its optimum pH
is 5.0 – 5.2. Narendranath and Power (2005) found that the optimum pH for
yeast growth and ethanol production by S. cerevisiae was pH 4.9. Limtong et al.
(2007) examined the ethanol production from sugar cane juice with 22% sugar
and found that the highest ethanol concentration was obtained when the
fermentation carried out at pH 5.0.
Table (4) : Effect of pH on ethanol production from beet molasses using S.cerevisiae
AU 71.
Alcohol %
Dry yeast weight (g/l)
Time
(hrs)
Ranges of pH
pH 5
pH 6
pH 7
pH 8
pH 5
pH 6
pH 7
pH 8
0
0.24
0.22
0.25
0.24
0
0
0
0
12
0.88
0.45
0.77
0.90
3.5
2.1
2.6
3.2
24
1.58
0.99
1.37
1.58
7.9
3.7
4.8
6.2
36
2.2
1.34
1.96
2.1
8.3
4.9
7.5
7.8
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48
2.85
1.55
pH 5 (dry weight)
2.11
2.52
9.2
8.1
8.4
3
pH 6 (dry weight)
8.9
14
pH 7 (dry weight)
pH 8 (dry weight)
12
2.5
pH 5 (alcoho%l)
pH 6 (alcoho%l)
10
pH 7 (alcoho%l)
pH 8 (alcoho%l)
Dry yeast weight (g/l)
8
1.5
6
Alcohol level %
2
1
4
0.5
2
0
0
0
12
24
36
48
Time(hours)
Figure (3): Effect of pH on dry yeast weight and ethanol production by S.cerevisiae
AU 71 grown on beet molasses at 20% sugar concentration and 30˚C
temperature for 48 hours on 120 rpm.
References
Bertolini M. C., Ernandes J. R and Laluce, C (1991): New yeast strains for
alcoholic fermentation at higher sugar concentration. Biotechnol Lett 13:
197-202.
Borzani, w. and Vario, M.L.R. (1958): Quantitative adsorption of methylene
blue by dead yeast cells. J. Bacteriology 76: 251 - 255.
Borzani, W., Garab A, Pires M H, Piplovic R and De Ia Higuera G. A
(1993): Batch ethanol fermentation of molasses: a correlation between the
time necessary to complete the fermentation and the initial concentrations
of sugar and yeast cells. World J Microbiol Biotechnol 9: 265-68.
European Community (2000): Community reference methods for the analysis
of spirit drinks. Off. J. Eur. Community Reg. Ec No 2870/000. December
10-13 November 2012, Aswan, Egypt
A. A. Zohri et al
P . 1/
10
International Conference on:
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19, Brussels.
Gough, S and McHale, A. P. (1998): Continuous ethanol production from
molasses at 45˚C using alginate immobilized Kluyveromyces marxianus
IMB3 in a continuous flow bioreactor. Bioprocess Engineering, 19: 3336.
Jones, A. M, Thomas K. C. and Inglew W. M., (1994): Ethanolic fermentation
of molasses and sugarcane juice using very high gravity technology. J.
Agric. Food Chem 42: 1242-1246.
Laluce C, Abud C J, Greenhalf W and Sanches Peres M F (1993):
Thermotolerance behaviour in sugarcane syrup fermentations of wild type
yeast strains selected under pressures of temperature, high sugar and
added ethanol. Biotechnol Lett 15: 609-14.
Limtong, S.; Sringiew, C. and youngmanitchai, W. (2007): Production of fuel
ethanol at high temperature from sugar cane juice by a newly isolated
Kluyveromyces marixianus. Bioresource Technology, 98: 3367-3374.
Maysa M.A. Ali., (2010): Studies on production of ethanol and single cell
proteins by local yeast strains, M.Sc. Thesis, Botany Department, Faculty
of Science, Assiut University, Assiut, Egypt.
Morimura S, Ling Z Y and Kida K (1997): Ethanol production by repeated
batch fermentation at high temperature in a molasses medium containing
a high concentration of total sugar by thermotolerant flocculating yeast
with improved salt tolerance. J Ferment Bioeng 83: 271-74.
Narendranath, N. V. and Power, R. (2005): Relationship between pH and
medium dissolved solids in terms of growth and metabolism of
lactobacilli and Saccharamyces cerevisiae during ethanol production.
Appl. Environ. Microbiol. 71: 2239-2243.
Pratt-Marshall, P. L.; Brey, S. E.; de Costa, S.; Bryce, J. H. and Stewart, G.
G. (2002): High gravity brewing – an inducer of yeast stress. Brewer̕ s
Guardian. 131: 22-26.
Pratt-Marshall, P. L.; Bryce, J. H. and Stewart, G. G. (2003): The effect of
osmotic pressure and ethanol on yeast viability and morphology. Journal
of the Institute of Brewing. 109: 218-228.
Prescott, S. C. and Dunn, (1959). Industrial Microbiology. Third Edition,
McGraw-Hill Book Company, Inc. New York, Toronto, London. 944.
Reddy, L. V. A. and Reddy, O. V. S. (2006): Rapid and enhanced production
of ethanol in very high gravity (VHG) sugar fermentation by
10-13 November 2012, Aswan, Egypt
A. A. Zohri et al
P . 1/
11
International Conference on:
“New Role for the World Sugar Economy in a Changed Political and
Economic Environment ”
Saccharomyces cerevisiae: Role of finger millet (Eleusine coracana L.)
flour. Process Biotechnology.
41: 726-729.
Russell, I. (2003): Understanding yeast fundamentals. In: Jacques KA, Lyons
TP, Kelsall DR, editors. The alcohol textbook-a reference for the
beverage, fuel and industrial alcohol industries. UK: Nottingham. P. 85119.
Singh D, Nigam P, Merchant R and Mchole A P (1998): Ethanol production at
elevated temperatures and alcohol concentration: Part 2- use of
Kluyveromyces maxianus IMB3. World J Microbiol Biotechnol 14: 82334.
Sols, A.; Gancedo, C. and Delafuente, G. (1971): The yeasts. In Rose, A. H.;
Harrison, J. S., editors, Vol. 2. London: Academic Press. 271.
Sree N K Sridhar M, Suresh K, Bharat I M and Rao L V (2000): High
alcohol production by repeated batch fermentation using immobilized
osmotolerant S.cerevisiae. J Indust Microbiol Biotechnol 24: 222-26.
Yadav BS, Sheoran, A., Rani, U. and Singh, D., (1997): High ethanol
productivity in an immobilized cell reactor Indian J Microbiol 37: 65-67.
Zohri, A. A. and Eman Mostafa., (2000): Ethanol production from dates in
Saudi Arabia on industrial scale. Microbiology, 28(2): 76-81.
10-13 November 2012, Aswan, Egypt
A. A. Zohri et al
P . 1/
12