INT J CURR SCI 2012, 219-228
RESEARCH ARTICLE
ISSN 2250-1770
Comparative study of continuous ethanol fermentation from molasses by using
Saccharomyces cervisiae and Schizosaccharomyces pombe
Hemamalini V*, S.G.E. Saraswathy, C. Hema and S. Geetha
Department of Plant Biology and Biotechnology, Arignar Anna Govt Arts College for Women, Walajapet, Tamil Nadu, India
*Corresponding author: E-mail:[email protected]; Phone: +91-9841425466
Abstract
Ethanol production has been carried out from molasses by yeast Saccharomyces cervisiae and Schizosaccharomyces
pombe. The level of alcohol production from molasses by the use of both these organisms in continuous ethanol fermentation.
The highest percentage of alcohol production by Saccharomyces cervisiae with specific gravity 1.032 was 7.20% whereas
Schizosaccharomyces pombe showed 5.25% of alcohol production with specific gravity 1.032. Batch fermentation was conducted
by these organism for the role of bio protease enzyme in ethanol production. Higher alcohol production was notiied in flask
which contained enzymes with Saccharomyces cervisiae and Schizosaccharomyces pombe. It was found that Saccharomyces
cervisiae with enzyme added batch fermentation showed 8% of alcohol production is higher compared with free cells as only 6%
of alcohol production. Amount of total reducing sugar and fermentation efficiency of the substrate molasses were calculated and
tabulated. Molasses is a complex substrate that has wide range of nutrients that are not often completely metabolized by the
microbial inoculums. Analysis of COD reduction rate of Saccharomyces cervisiae used waste water was 41.50%. Analysis of
COD reduction rate of Schizosaccharomyces pombe used waste water was 50.78%.
From this study, we conclude that
Saccharomyces cervisiae yielded better results in alcohol production compared to Schizosaccharomyces pombe.
Keywords: Saccharomyces cervisiae, Schizosaccharomyces pombe, ethanol fermentation, molasses
Received: 19th December; Revised: 29th December; Accepted: 30thJanuary; © IJCS New Liberty Group 2012
renewable sources gives no net contribution to “Green
Introduction
Microorganisms play an important role in
gases” vehicles that turn straight alcohol are cleaner with
biotransformation of waste products into human, animal
respect to emission of hydrocarbon, carbon monoxide and
and plant consumables. Yeast cells are used in household
one better to drive in many aspects (Wyman and Good
fermentation, food production, industrial fermentation and
man, 1993).
biotransformation process. Fermentation is a process in
Ethanol had been trusted as an alternate fuel for
which complex molecules are converted into simple sugar
the further utilization of a world-wide interest in the
by the role of microorganisms (Patel, 1991). Ethanol is an
utilization of bio-ethanol as an energy source which in turn
important industrial solvent and chemical feed stock for the
stimulate studies on the cost and efficiency of fermentative
synthesis
adhesives,
organisms. Cheapest fermentation substrates and optimum
plastics, plasticizers and host for other chemicals (Prescott
environmental conditions are needed for fermentation.
and Dunn, 1959). It is also an energy feed stock and serve
(Gunasekaran and Chandraraj, 1999). The main raw
as a fuel in internal combustion and possible future decline
material used in India for the ethanol production is
in oil supplies are stimulating more use of ethanol as an
sugarcane molasses. The demand for ethanol is also
octane enhancer in unblended gasoline and as a gasoline
increasing day by day. The prerequisite for achieving
substitute (Kiem and Venkatasubramanian, 1989). Today
process improvement in alcohol production depends on the
alcohol technology is again reviving the production of
selection of suitable yeast strains for high ethanol yield to
alcohol as a fuel in being given attention with the world
maximize substrate utilization, minimize fermentation
energy crisis (Jogdand, 1993). It has enhanced interest in
capital costs and reduce ethanol recovery costs. Molasses is
of
pharmaceuticals,
detergents,
Hemamalini et al., 2012
dark coloured syrup left after extraction of sugar from
gravity works and tolerance to high concentration of
sugarcane. It contains about 62% of carbohydrates in the
ethanol. Protease is one of the most important industrial
form of 30% uncrystalized sucrose and about 32% of invert
enzymes accounting for 60% of total worldwide enzyme
sugar which is a mixture of glucose and fructose (Patel,
scale. The major market for enzyme is Western Europe
1999). The sugar, which is converted into molasses, is
and is found to be the emerging market. In the next fifteen
adjusted to 14-16%, which permits an alcohol content of 8-
years, Asia is expected to account for nearly 20% of the
10 volume percentage in the fermented wort. (Krotzschmar,
global enzyme market. The leading enzyme marketers in
1995). Ethanol production of fuel has been industrialized in
India are Biocon Pvt. Ltd., Advanced Bio Chemicals Ltd.,
a number of developing countries. Saccharomyces spp. has
Maps industries; Textan chemicals; Anil starch products
been used to ferment molasses for ethanol production,
Ltd., Maize products and SPIC Ltd. Ethanol production has
tolerance of these strains to high alcohol levels and sugar
been carried out from molasses by yeast Saccharomyces
concentration limits the concentration of alcohol that can be
cervisiae and Schizosaccharomyces pombe.
obtained from such process. (Ezeogu and Okolo, 1994).

Continuous ethanol fermentation from molasses by the
Saccharomyces cervisiae is a high ethanol yielding yeast
use of various yeast culture such as Saccharomyces
which was used for ethanol production from the ultra
cervisiae and Schizosaccharomyces pombe
filtrates of whey that was hydrolyzed first to give glucose

and glactose (Roland and Alm, 1975).
Batch fermentation was conducted by these various
yeast with Bio-protease enzyme
Saccharomyces cervisiae were examined for their

Analysis of molasses medium
production ability in molasses than tapioca and sorghum. It

Find out and calculate the COD reduction rate in spent
gave high efficiency of ethanol production by using
wash and recycle sample.
substates like sorghum, tapioca (Brown and Oliver, 1982).

Collection of scum sample from fermentor.
Schizosaccharomyces pombe is fission yeast, which has

Effect of Bio-protease enzyme
high growth rate increased fermentation ratio, high ethanol
Materials and methods
tolerance
Collection of sample and source of microbial strains
temperature
tolerance,
and
osmotolerent.
Schizosaccharomyces pombe produced maximum bimass.
Molasses sample was collected from the distillery
Traditionally ethanol is produced from cane molasses by
unit of Dharani Sugars and Chemicals Pvt. Ltd.,
fermentation with yeasts. Due to product inhibition ethanol
Vasudevanallur,
concentration is usually limited to 8-9% by volume
producing
(Heraldson and Bjorling, 1981). Ethanol fermentation is a
Schizosaccharomyces pombe were obtained from microbial
continuous process, the molasses flow in and fermented
type culture collection (MTCC), Chandigarh (India).
Thirunelveli
strains
district.
Saccharomyces
The ethanol
cervisiae
and
wash flows out of the fermentor. The concentration of yeast
cell cycle can be segregated in different fermentors for the
Maintanance of culture
yeast cell growth and carbon dioxide evolved. The process
Saccharomyces cervisiae was routinely sub
is continued and yeast cells remain in suspension. Finally
cultured and maintained in MGYP agar slant at every 15
the yeast cells are removed and clear wash is taken for
days intervals and stored at 4C.
distillation. The yeast strains normally employed in
pombe was subcultured and maintained in molasses agar
industrial process show a limited tolerance to ethanol,
slants.
temperature and high osmotic pressure of the medium
Inoculum preparations for ethanol production
Schizosaccharomyces
(Casey and lngledew, 1986; Amore et al., 1990; Bertolini
The yeast inoculum was prepared (Nellaiah and
et al., 1991). An important aspect in ethanol production, in
Gunasekaran, 1991). To a 50 ml of sterile molasses
search of new strains succeeding in industry depends upon
medium and glucose medium, a loop full of yeast
the characters like thermo tolerance, capacity to grow in
Schizosaccharomyces pombe and Saccharomyces cervisiae
Hemamalini et al., 2012
from fresh slant culture were inoculated separately. The
Batch fermentation
inoculum was incubated at room temperature for 24 hrs.
Bio protease enzyme was added with molasses
Continuous ethanol fermentation
medium for ethanol fermentation.
Molasses as the
Continuous ethanol fermentation was done in 500
substrate was prepared by diluting with distilled water,
ml molasses medium, which was prepared at 1.032 brix
until 1.090 brix gravity of sugar concentration had reached.
specific gravity, autoclaved and cooled. Yeast inoculums
The pH was adjusted as 4.5, 5.0 and 5.5.
Schizosaccharomyces pombe and Saccharomyces cervisiae
concentration (15 to 18%) and 10 ppm of our enzyme
was inoculated into separate flasks. Incubate the flasks for
solution (0.5 g) were added. To this fermentation broth
8 hrs at 37C and thus yeast was grown. The fermentation
10% of yeast inoculums Saccharomyces cervisiae was
of foam indicates the growth of the yeast cells. Take
inoculated. The same procedure was followed in another
another 1500 ml molasses medium, which was prepared at
flask and inoculated with 10% of yeast inoculums
1.032 brix specific gravity and sterilized. Add this molasses
Schizosaccharomyces pombe. It was incubated after room
medium into the yeast inoculums and the total volume of
temperature 37C for 48 hrs. After incubation period the RS,
˚
The sugar
2000 ml culture medium was incubated for 8 hrs at 37 C.
alcohol percentage and TVA was analyzed. In the same
After 8 hrs, the total 2000 ml of culture medium was added
manner two control flask were also run without adding bio
to 2500 ml of freshly prepared molasses medium with1.032
protease enzyme.
brix specific gravity and incubated for 8 hrs at 37C. The
Estimation of residual sugar (RS)
total amount of culture medium is 4500 ml. After 8 hrs 250
Incubate 20% of fermented wash taken for
ml fermentated wash was taken from the flask and check
titration. 5 ml of Fehling’s solution A and B with 100 ml
the specific gravity of the sample i.e. called initial gravity.
of distilled water were taken in a 250 ml conical flask and
Gravity was highly decreased and then 100 ml
was taken and discarded from the flask.
Then freshly
heated. Few drops of methylene Blue indicator were added
while heating. The solution turned blue in colour and the
prepared 100 ml molasses medium with high specific
titration continued till brick red colour.
gravity (1.070 brix specific gravity) was added to the flask
Calculation
culture. After every one hour the specific gravity of the
Reducing sugar = 27.2 / titre value
i.e. 27.2 – Fehling’s factor value.
culture medium was analyzed and the same procedure was
followed every one hour. After every four hours residual
Estimation of alcohol content
sugar level, alcohol percentage and total volatile acid were
noted.
The fermented wash (250 ml) was taken and
The same procedure was continued for 5 days.
made up to 500 ml with distilled water. The sample was
Alcohol percentage depends upon the reducing sugar level,
transferred into the round bottom flask and allowed for
if the sugar level decreases, the alcohol percentage
condensation. Then 250 ml of condensate was collected
increases. Sugar was converted into ethanol by the action
and temperature of the condensate was noted. The
of yeast cultures such as (Schizosaccharomyces pombe and
condensate was poured into the measuring jar. The alcohol
Saccharomyces cervisiae). When sugar was present in high
content was estimated by a hydrometer which is designed
level the organism utilizes the sugar slowly resulting in
for measuring only alcohol percentage.
production of ethanol. According to this procedure, alcohol
Calculation
percentage and residual sugar levels were noted. At the
100-alcohol percentage / 1.66 (alcohol standard
end of the experiment, as the sugar level has decreased the
value)
alcohol percentage had increased to a higher level. The
Estimation of total volatile acids (TVA)
specific gravity of the solution was set up as 1.032, 1.070
and 1.090 etc.
Fermented wash 100 ml and 100 ml distilled
water was mixed with 5 ml of sulphuric acid (50%). This
Hemamalini et al., 2012
mixture was taken in a round bottom flask and distillated
The titration was continued till the blue colour disappeared
using distillation unit. Exactly collected 150 ml distillate
and there was an appearance of permanent brick red, which
and transferred the distillate into the conical flask. To this
was noted as the end point.
added 5 drops phenolphthalein as an indicator. The burette
Calculation
was filled with Sodium Hydroxide solution (0.5N).
5.128 / FF x TV x DF
Sodium hydroxide was titrated against distillate with
25.64 / 26 = 0.986
indicator until the pale pink colour appears. The end point
Estimation fermentation efficacy
(pale pink colour) was noted.
Calculation
Total Volatile Acid = Titer value X 857.14
Analysis of fermentation medium
Biological conversion of carbohydrate residue
50 gm of molasses was taken and mixed with
water. Added two grams of di-ammonium Phosphate and
adjusted the pH of the flask to 4.5 with dilute H2SO4.
Made up the volume up to 300 ml. Sterilized the flask at 15
lbs pressure for about 10 min. After sterilization cool the
mainly involves traditional fermentation processes.
flask at room temperature. Added 2 gm of yeast to the flask
Estimation of total reducing sugar
and kept in the incubator at 37C for 48 hrs. Find out the
5 gm of molasses was dissolved in 100 ml of
specific gravity by specific gravity method and determined
distilled water and 5 ml of concentrated hydrochloric acid
the initial total reducing sugar. Fermented wash 250 ml was
was added. Then it was boiled at 70C and cooled. The
taken and added with 250 ml of distilled water.
solution was neutralized by the addition of 6 N Sodium
mixture was taken in a round bottom flask and distilled.
hydroxide until the red litmus paper turned to blue. The
250 ml of distillate corrections are applied, and the alcohol
neutralized solution was made up to 1000 ml taken in the
percentage was calculated from the standard table.
burette solution and titrated against 5 ml of both Fehling’s
A&B solution till wine red developed. 4 drops of
Fermentable sugar (%) = Total Reducing sugar –
Unfermentable sugar.
methylene blue were added as an indicator. The titration
Theoretical yield:
was continued till the blue colour disappeared and there
C12H22O11 ------ 2C6H12O6
was an appearance of permanent brick red, which was
C6H12O6
noted as the end point.
Calculation
Total Reducing Sugar = 5.128 / TV x FF x DF
FF – Fehling’s Factor, TV – Titre Value, DF –
Dilution Factor (0.005)
25.64 / 26.5 = 0.967
Estimation of unfermentable sugar
25 gm of raw molasses was weighed and mixed
This
------ 2C2H5OH + 2CO2
From this molecular formula, 100 gm of sugar
gives 64.4 ml of ethanol. 100 gm of sugar = 64.4 ml of
alcohol.
Theoritical yield – Fermentable Sugars x 64.4
Calculation
Fermentation Efficacy (%) – Actual Percentage
of alcohol / Theoritical yield x 100
Bioenergy potential of distillery effluent
with 25 gm of dry yeast. This mixture was made up to 150
There are 285 distilleries in India producing 2.7
ml with ordinary water. Add a few grams of urea and di-
billion litres of alcohol generating 40 billion litres of waste
ammonium phosphate. Allow it to ferment for 24 hrs.
water annually. The enormous distillery waste water has
After fermentation, the 150 ml was made up to 250 ml, 100
potential to produce 1100 milion cubic meters of biogas.
ml of tabletop centrifuge. Yeast sludge was precipitated.
Distillery waste water known as spent wash. According to
The 50ml of supernentant was taken and make up to 100
a recent estimate, the alcohol production in India has
ml. It was taken in the burette solution and titrated against
reached the 27 million litre mark. The proportions of waste
5 ml of both Fehling’s A&B solution till wine red colour
water, generally known as spend wash, in nearly 15 times
developed. A few drops of methylene blue was added.
the total alcohol production. This massive quantity,
Hemamalini et al., 2012
approximately 40 billion litres of effluent, if disposed
was taken and made up to 500 ml (Dilution factor for spent
untreated can cause considerable stress on the water causes
wash = 50). (Dilution factor for recycle = 25). From that 1
leading to widespread damage to aquatic life. Bio-gas
ml of recycle sample was taken and made up to 250 ml.
normally 60% methane gases which is a well recognized
Diluted solutions were taken in COD flask. An ounce of
fuel gas with minimum air pollution potential. This is more
mercuric sulphate was added. Then 5ml of potassium
significant in Indian context and many distilleries are
dichromate is added and 15 ml of concentrated H2SO4 was
adopting this technology.
added in the flask. It was cooled in water and it was
Composition of spent wash
refluxed for two hours in heating mantle. Then it was left to
Spent wash is characterized by its colour, high
cool down and totrated against 0.1N FAS solution. Ferroin
temperature, low pH, high ash content and contains high
was used as an indicator. End point was the appearance of
percentage of dissolved organic and inorganic matter of
brick red colour. Ins blank flask was also kept along this.
which 50% may be present as reducing sugars. It contains
Calculation
about 90 – 93% water and 7 – 10% solids; sugar being 2 -
Blank – sample x Normality of FAS x DF x 8000
20% and protein 10 – 11% in the dry spent wash. Indian
/ 20 = mg / 1
spent wash contain very high amounts of potassium,
Dilution factor
calcium chloride, sulphate and BOD (around 50,000 mg/lt)
For spent wash
- 500
when compared to spent wash in other countries.
For recycle
- 250
Biomethanation plant
COD reduction rate
Spent wash is used as a feed for Bio-gas plant. In
Calculation
Dharani Sugar anaerobic digester contain plastic media in
Spent wash – recycle / spent wash x 100
the centre of the digester tank. This is used for microbes to
Estimation of total volatile acid inspent wash and recycle
stick on media in the centre of the digester tank. This is
samples
used for microbes to stick on media surface and active for
A mixture of 100 ml sample and 100 ml distilled
long time to produce bio-gas. Methanogenic and acetogenic
water were taken and mixed with 5 ml of sulphuric acid
bacteria are used for bio gas production. The top of the
(50%). This mixture was distilled in distillation unit.
plant space called Gas zone. Recirculation pump was
Exactly, 150 ml of distillate was collected and five drops of
present at the bottom of the tank which is used for even
phenolphthalein as an indicator was added. 0.5 N sodium
mixing of feed and bacteria. The capacity of the digester is
hydroxide was taken in burrete and titrated against distillate
3
4000 m (40,00,000). Microbiological processes involved
with indicator till pale pink colour appeared. The end point
in anaerobic digestion centre around Methanogenic bacteria
was noted.
differ significantly from acid forming bacteria in terms of
Calculation
physiology; nutritional requirements and sensitivity to
formers are separated.
enhanced.
The efficacy of the system is
The methane forming bacteria could be
effectively and eliminating the potential problems before
the methane forming bacteria are subjected to stress. Post
methanation wastewater is called Recycle. If used carefully
for irrigation of agricultural crops can produce more than
30,000 tonnes of bio mass annually.
Estimation of chemical oxygen demand
100 ml of sample (spent wash/recycle) was taken
and centrifuged.
Titre value x 0.5 x 1000 / 70s
From that 1 ml of solvent wash sample
Results and discussion
Saccharomyces
cerivisiae
and
Schizosaccharomyces pombe are promising strains for the
ethanol production that are actively researched worldwide.
Saccharomyces cervisiae is used all over the world as the
major ethanol producing organism. Industrial usefulness of
Schizosaccharomyces pombe was also recorded by Gomes
et al. (2002), where this strain was recorded to have
osmotolerant, alcohol tolerance and sustained alcohol
Hemamalini et al., 2012
production. The continuous ethanol fermentation by both
250ml
1.085
1.035
these organisms was reported in table 1 and 2.
250ml
1.085
1.036
250ml
1.085
1.036
Table 1. Continuous ethanol production by Sacharomyce
250ml
1.085
1.037
cervisiae
250ml
1.085
1.037
250ml
1.085
1.039
250ml
1.085
1.039
250ml
1.085
1.038
250ml
1.085
1.040
250ml
1.085
1.038
250ml
1.085
1.039
250ml
1.085
1.041
250ml
1.085
1.040
250ml
1.085
1.040
250ml
1.085
1.039
250ml
1.085
1.038
250ml
1.085
1.038
250ml
1.085
1.037
250ml
1.085
1.038
250ml
1.085
1.040
250ml
1.085
1.039
250ml
1.085
1.037
250ml
1.085
1.036
250ml
1.085
1.038
250ml
1.085
1.036
250ml
1.085
1.037
250ml
1.085
1.036
250ml
1.080
1.035
250ml
1.080
1.034
250ml
1.080
1.036
250ml
1.080
1.034
250ml
1.080
1.033
250ml
1.080
1.035
250ml
1.080
1.034
250ml
1.080
1.033
250ml
1.080
1.031
250ml
1.080
1.030
250ml
1.070
1.032
250ml
1.070
1.031
250ml
1.070
1.033
250ml
1.070
1.030
250ml
1.070
1.029
250ml
1.070
1.028
250ml
1.070
1.027
250ml
1.070
1.029
250ml
1.070
1.028
Substrate
Added
Setup
Gravity
Initial
Gravity
100ml
1.070
1.012
100ml
1.070
1.013
100ml
1.070
1.013
100ml
1.070
1.015
250ml
1.080
1.016
250ml
1.080
1.019
250ml
1.080
1.021
250ml
1.080
1.024
250ml
1.080
1.022
250ml
1.080
1.027
250ml
1.080
1.026
250ml
1.080
1.029
250ml
1.080
1.030
250ml
1.080
1.030
250ml
1.080
1.033
250ml
1.080
1.032
250ml
1.080
1.030
250ml
1.080
1.031
250ml
1.080
1.033
250ml
1.080
1.034
250ml
1.080
1.034
250ml
1.080
1.030
250ml
1.080
1.029
250ml
1.080
1.028
250ml
1.080
1.029
250ml
1.080
1.029
250ml
1.080
1.030
250ml
1.080
1.028
250ml
1.080
1.026
250ml
1.080
1.027
250ml
1.080
1.028
250ml
1.080
1.027
250ml
1.080
1.029
250ml
1.080
1.031
250ml
1.080
1.030
250ml
1.085
1.032
250ml
1.085
1.032
250ml
1.085
1.033
Alcohol
%
2.67%
2.9%
3.1%
3.45%
3.0%
2.86%
3.06%
Reducing
Sugar
1.20
1.28
1.36
1.69
1.98
2.01
1.97
3.56%
1.81
3.82%
1.76
4.03%
1.71
4.24%
1.66
4.9%
1.60
5.3%
1.45
5.8%
1.42
6.2%
1.39
6.92%
1.36
Hemamalini et al., 2012
250ml
1.070
1.027
250ml
1.070
1.030
250ml
1.070
1.029
250ml
1.070
1.028
250ml
1.070
1.029
250ml
1.070
1.025
250ml
1.070
1.024
250ml
1.070
1.026
250ml
1.070
1.022
250ml
1.070
1.023
250ml
1.070
1.023
250ml
1.070
1.024
250ml
1.070
1.022
250ml
1.070
1.021
250ml
1.070
1.023
250ml
1.070
1.022
Table
2.
Continuous
ethanol
7.02%
7.15%
1.31
1.29
7.17%
1.25
7.20%
1.21
production
by
Schizosaccharomyces pombe
Substrate
Added
100ml
100ml
100ml
100ml
250ml
250ml
250ml
250ml
250ml
250ml
250ml
250ml
250ml
250ml
250ml
250ml
250ml
250ml
250ml
250ml
250ml
250ml
250ml
250ml
250ml
250ml
250ml
250ml
250ml
250ml
250ml
Setup
Gravity
1.070
1.070
1.070
1.070
1.080
1.080
1.080
1.080
1.085
1.085
1.080
1.080
1.080
1.080
1.080
1.085
1.085
1.085
1.085
1.085
1.085
1.085
1.085
1.085
1.065
1.065
1.065
1.065
1.065
1.065
1.065
Initial
Gravity
1.015
1.015
1.016
1.016
1.018
1.019
1.022
1.023
1.025
1.028
1.030
1.031
1.033
1.034
1.036
1.037
1.037
1.038
1.040
1.041
1.041
1.042
1.043
1.043
1.044
1.043
1.044
1.043
1.042
1.041
1.039
Alcohol
%
2.05%
1.92%
3.19%
2.65%
4.88%
4.22%
Reducing
Sugar
1.20
1.91
3.24
2.61
2.69
2.47
250ml
250ml
250ml
250ml
250ml
250ml
250ml
250ml
250ml
250ml
250ml
250ml
250ml
250ml
250ml
250ml
250ml
250ml
250ml
250ml
250ml
250ml
250ml
250ml
250ml
250ml
250ml
250ml
250ml
250ml
250ml
250ml
250ml
250ml
250ml
250ml
250ml
250ml
250ml
250ml
250ml
250ml
250ml
250ml
250ml
250ml
250ml
250ml
250ml
250ml
250ml
250ml
1.065
1.065
1.065
1.060
1.060
1.060
1.060
1.060
1.060
1.060
1.060
1.060
1.060
1.060
1.060
1.060
1.060
1.060
1.060
1.060
1.060
1.060
1.060
1.060
1.060
1.060
1.060
1.060
1.060
1.060
1.060
1.060
1.060
1.060
1.060
1.060
1.060
1.060
1.060
1.060
1.060
1.060
1.060
1.060
1.070
1.070
1.070
1.070
1.070
1.070
1.070
1.070
1.036
1.037
1.039
1.038
1.038
1.037
1.036
1.035
1.035
1.035
1.034
1.034
1.033
1.032
1.032
1.031
1.031
1.030
1.032
1.031
1.030
1.030
1.030
1.029
1.030
1.030
1.031
1.030
1.030
1.030
1.030
1.029
1.030
1.030
1.030
1.030
1.030
1.030
1.030
1.028
1.029
1.029
1.026
1.029
1.028
1.030
1.029
1.030
1.030
1.029
1.028
1.029
250ml
250ml
250ml
250ml
250ml
250ml
250ml
1.070
1.070
1.070
1.070
1.070
1.070
1.070
1.029
1.030
1.031
1.031
1.030
1.030
1.029
4.03%
2.21
4.39%
2.69
4.56%
2.17
5.06%
1.68
5.40%
1.39
4.88%
1.73
4.33%
1.66
4.64%
1.51
4.58%
1.29
4.75%
1.66
4.86%
1.74
5.00%
1.36
Hemamalini et al., 2012
250ml
250ml
250ml
250ml
250ml
250ml
250ml
250ml
250ml
250ml
1.070
1.070
1.070
1.070
1.070
1.070
1.070
1.070
1.070
1.070
1.029
1.030
1.030
1.030
1.030
1.031
1.031
1.032
1.030
1.029
also maintained without protease enzyme by using
molasses as the fermentation substract. The RS, TVA and
alcohol percentage were noted for the both, enzyme added
5.20%
1.40
flask as well as the free cells contained flask.
Table 4. Analysis of fermentation medium molasses
Factors
Percentage
TRS
49.915%
UFS
4.60%
On this basis Saccharomyces cervisiae and
FS
45.31%
Schizosaccharomyces pombe were employed individually
AIC
4.96%
to understand their efficacy in ethanol fermentation. The
FE
51.01%
highest
percentage
of
5.25%
alcohol
1.37
production
by
Saccharomyces cervisiae with specific gravity 1. 032 was
TRS – Total Reducing Sugar
7.20%, whereas Schizosaccharomyces pombe showed
UFS – Unfermentable Sugar
5.25% of alcohol production with specific gravity 1.032
FS – Fermentable Sugar
(Mandeep Kawr and Kocher, 2002). Batch fermentation
AIC - Alcohol
results were noted and tabulated in table 3 and 3A.
FE – Fermentation efficiency
Table 3. Batch fermentation by Saccharomyces cervisiae
with enzyme and without enzyme
Factor
Initial
Concentration of bio protease enzyme 5 mg was
added with 100 ml molasses medium with 1.090 specific
Control
Enzyme
gravity. From our result, alcohol production was enhanced
Added
only in the enzyme added flask. The control flasks
produced alcohol in lesser amount, when compared to the
1,090
1.090
Final gravity
1.044
1.032
Alcohol
6.24
8.0
Saccharomyces cervisiae with enzyme containing flask and
RS
1.79
1.11
Schizosaccharomyces pombe with enzyme containing flask.
TVA
2214.217
3599.983
Table 5. Analysis of COD reduction rate in Saccharomyces
gravity
experimental flask. Higher alcohol production was notified
between
various
type
of
yeast
strains
such
as
cervisiae used waste water
Table 3 A. Batch fermentation by Schizosaccharomyces
Control
Enzyme
Blank
24.3
-
added
Spent Wash
19
1,06,000
Recycle
18-1
62,000
pombe with enzyme and without enzyme
Factor
Control
Enzyme
Factor
Initial gravity
1,090
1.090
Final gravity
1.52
1.042
Alcohol
5.00
6.74
RS
2.00
1.67
TVA
1571.422
2342.85
COD reduction
added
- 41.50%
The yeast Saccharomyces cervisiae was highly
fermentative when it was added with enzyme. It produces
better
result
for
ethanol
production
than
For the study of role of protease enzyme in
Schizosaccharomyces pombe. It was concluded that
ethanol production, the batch fermentation of ethanol was
Saccharomyces cervisiae with enzyme added batch
carried out with protease enzyme. The control flask was
fermentation showed 80% of alcohol production, as higher
Hemamalini et al., 2012
when compared with free cells as only 6% of alcohol
pombe.
production (Heraldson and Bjorling, 1981).
Schizosaccharomyces pombe were used as free cells in
Table
5a.
Analysis
of
COD
reduction
rate
in
Schizosaccharomyces pombe used waste water
The
yeasts
Saccharomyces
cervisiae
and
continuous ethanol fermentation and the ethanol yield was
noted as 5.25% and 7.20% respectively. The Bio-protease
enzyme, obtained from Biocon enhanced the ethanol yield.
Enzyme
In batch fermentation, Saccharomyces cervisiae and
added
Schizosaccharomyces pombe was added with enzyme,
24.3
-
yielded higher ethanol than they were used as free cells
17.9
1,25,000
18
63,000
Factor
Control
Blank
Spent
Wash
Recycle
(6.74% and 8.0%). When yeast inoculums Saccharomyces
cervisiae and Schizosaccharomyces pombe were used as
free cells, the ethanol yield was 5.0% and 6.24%
respectively. The above indications emphasize that the use
COD reduction
of Schizosaccharomyces pombe was not efficient when
- 50.78%
Amount of total reducing sugar and fermentation
compared to Saccharomyces cervisiae. These two strains
efficiency of the substrate molasses were calculated and
were highly efficient when added with enzymes. However,
tabulated in table 4. Molasses is a complex substrate that
Saccharomyces cervisiae is a highly efficient fermentative
has wide range of nutrients that are not often completely
organism.
metabolized by the microbial inoculums.
Saccharomyces cervisiae yielded better result in ethanol
Attempts are
being made throughout the world to increase ethanol
From
this
study,
we
concluded
that
production than Schizosaccharomyces pombe.
production by making the substrates more metabolizable. In
this idea protease enzyme was added to simplify these
complex molecules. Industrial exploitation of renewable
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