AMERICAN JOURNAL OF SCIENTIFIC AND INDUSTRIAL RESEARCH

© 2011, Science Huβ, http://www.scihub.org/AJSIR

ISSN: 2153-649X doi:10.5251/ajsir.2011.2.4.694.706

Utilization of sugar refinery waste (molasses) for ethanol production-

using Saccharomyces Cervicae
O. A. Osunkoya, N. J. Okwudinka
Department of Chemical Engineering, Covenant University, Ota, Ogun State, Nigeria
ABSTRACT
An analysis of the current situation and perspective on biomass-to-ethanol is provided in this
study. Various conversion pathways are compared from technical, economic, and environmental
points of view. This study also deals mainly with the yield of ethanol from molasses with respect
to the dilution ratio and the amount of yeast used for fermentation keeping the temperature and
fermentation duration constant. After the study it was observed that with an increase in yeast
quantity the ethanol yield decreases in a fluctuating manner until the quantity of yeast is about
grams it begins to increase again with the action of the yeast greatly depending on the dilution
ratio.
Keywords: Ethanol; Molasses; Sugar Refinery waste; Saccharomyces Cervicae;

INTRODUCTION It also would help in further research areas such as

biodegradation of waste products as molasses is a
Liquid bio-fuels are receiving increasing attention waste product after the refining of sugar.
worldwide as a result of the growing concerns about
oil security of supply and global climate change. In ETHANOL
most developing countries, the emerging bio-fuels
industry is perceived as an opportunity to enhance
economic growth and create or maintain jobs,
particularly in rural areas. The liquid bio-fuels market
is shared mainly between bio ethanol and biodiesel,
with more than 85% market share for the former in
2005. The main advantage of bio ethanol is the
possibility to blend it in low proportions with gasoline
(5 to 25% bio ethanol by volume) for use, without any
significant change, in internal combustion engines. (1) Fig 2. 1: Chemical Structure of Ethanol

Aims and objective of study:
The aim of this project is to study the dilution rate of
molasses and the effect of yeast on the yield of
ethanol using batch fermentation.
METHOD AND SCOPE OF STUDY
Various methods can be used for the specific
purpose of the production of ethanol; this includes
Ethylene hydration, Fermentation, Cellulosic ethanol.
The above stated methods are pathways to ethanol
production but fermentation is applied in this paper.
RELEVANCE OF STUDY
This study poses great opportunities for the chemical
engineering profession in Covenant University and
Nigeria at large.
Ethanol, also called ethyl alcohol, pure alcohol, grain
alcohol, or drinking alcohol, is a volatile, flammable,
colourless liquid. In common usage, it is often
referred to simply as alcohol or spirits. Ethanol is a
straight-chain alcohol, and its molecular formula is
C2H5OH. Its empirical formula is C2H6O. An
alternative notation is CH3-CH2-OH, which indicates
that the carbon of a methyl group (CH3-) is attached
to the carbon of a methylene group (-CH2-), which is
attached to the oxygen of a hydroxyl group (-OH). It
is a constitutional isomer of di-methyl ether. Ethanol
is often abbreviated as EtOH, using the common
organic chemistry notation of representing the ethyl
group (C2H5) with Et.
Ethanol burning with its spectrum depicted. (5)
Hydrogen bonding causes pure ethanol to be
hygroscopic to the extent that it readily absorbs water
from the air. The polar nature of the hydroxyl group

causes ethanol to dissolve many ionic compounds,
notably sodium and potassium hydroxides,
magnesium chloride, calcium chloride, ammonium
chloride, ammonium bromide, and sodium bromide.(4)
Sodium and potassium chlorides are slightly soluble
in ethanol.(4) Because the ethanol molecule also has
a non polar end, it will also dissolve non-polar
substances, including most essential oils (6) and
numerous flavoring, coloring, and medicinal agents.
The addition of even a few percent of ethanol to
water sharply reduces the surface tension of water.
This property partially explains the “tears of wine”
phenomenon. Mixtures of ethanol and water that
contain more than about 50% ethanol are flammable
and easily ignited.
Pathways to ethanol production include:
ETHYLENE (ETHENE) HYDRATION
Ethanol is manufactured by reacting ethene with
steam. The reaction is reversible, and the formation
of the ethanol is exothermic.
Only 5% of the ethylene is converted into ethanol at
each pass through the reactor. By removing the
ethanol from the equilibrium mixture and recycling the
ethene, it is possible to achieve an overall 95%
conversion. (7)
THE MECHANISM FOR THE ACID CATALYSED
HYDRATION OF ETHENE (7)
Step 1
All of the hydrogen atoms in the phosphoric (V) acid
are fairly positively charged because they are
attached to a very electronegative oxygen atom.
One of these hydrogen’s is strongly attracted to the
carbon-carbon double bond.
The pi part of the bond breaks and the electrons in it
move down to make a new bond with the hydrogen
atom. That forces the electrons in the hydrogen-
oxygen bond down entirely onto the oxygen.
Step 2
The carbocation (carbonium ion) formed reacts with
one of the lone pairs on a water molecule. A
carbocation is one which carries a positive charge on
a carbon atom. (7)
Step 3
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Am. J. Sci. Ind. Res., 2011, 2(4): 694-706
Finally, one of the hydrogens on the oxygen is
removed by reaction with the dihydrogenphosphate
(V) ion, H2PO4-, formed in the first step.
The reversibility of this reaction (7)
This reaction is entirely reversible - and so each step
is reversible. In the reverse direction, this is a
dehydration of ethanol to make ethene.
Equations of reaction
C2H4 (g) + H2O (g) → CH3CH2OH (l).-------------Equation
2.1
C2H4 + H2PO4 → CH3CH2PO4H ---------------Equation
2.2
Fig 2 . 2: Ethylene hydration using phosphoric acid
catalyst
CH3CH2PO4H + H2O → CH3CH2OH + H2PO4-----------
Equation 2.3
Fig 2.3: Phosphoric acid catalyst regeneration
Fermentation
The breakdown of carbohydrates to ethanol, carbon
di-oxide and water using micro organisms. (10)
The many and varied raw materials used in the
manufacture of ethanol via fermentation are
conveniently classified under three types of
agricultural raw materials (11):
1. Sugar
2. Starches
3. Cellulose materials.
Fermentation from Sugars: Sugars from sugar
cane, sugar beets, molasses, and fruits) can be
converted to ethanol directly.
The most widely used sugar for ethanol fermentation
is blackstrap molasses which contains about 35 – 40
wt% sucrose, 15 – 20 wt% invert sugars such as
glucose and fructose, and 28 – 35 wt% of non-sugar
solids. Blackstrap (syrup) is collected as a by-product
of cane sugar manufacture. The molasses is diluted

to a mash containing 10 –20 wt% sugar. After the pH
of the mash is adjusted to about 4 – 5 with mineral
acid, it is inoculated with the yeast, and the
fermentation is carried out non-aseptically at 20 –
32°C for about 1 – 3days. The fermented beer, which
typically contains 6 – 10 wt% ethanol, is then set to
the product recovery in purification section of the
plant.
Fermentation from Starches: All potable alcohol
and most fermentation industrial alcohol are currently
made principally from grains. Fermentation of starch
from grain is somewhat more complex than
fermentation of sugars because starch must first be
converted to sugar and then to ethanol (12). Starch is
converted enzymatically to glucose either by diastase
presents in sprouting grain or by fungal amylase. The
resulting dextrose is fermented to ethanol with the aid
of yeast producing CO2 as co-product. A second co-
product of unfermented starch, fiber, protein and ash
known as distillers grain (a high protein cattle feed) is
also produced.
Fermentation from Cellulosic Materials: Each step
in the process of the conversion of cellulose to
ethanol proceeded with 100% yield; almost two-thirds
of the mass would disappear during the sequence,
most of it as carbon dioxide in the fermentation of
glucose to ethanol. This amount of carbon dioxide
leads to a disposal problem rather than to a raw
material credit. Another problem is that the aqueous
acid used to hydrolyze the cellulose in wood to
Table 2. 1 : Bacteria useful for fermentation
Mesophilic Organisms
Clostridium sporogenes
Clostridium indolis
(pathogenic)
Clostridium sphenoides
Clostridium sordelli
(pathogenic)
Zymomonas mobilis
(syn.Anaerobica) (anaerobe)
Zymomonas mobilis
Ssp. Pomaceas
Spirochaeta aurantia
Spirochaeta stenostrepta
Spirochaeta litoralis
Erwinia amylovora
Leuconostoc mesenteroides
Streptococcus lactis
Sarcina ventriculi
(syn. Zymosarcina)
Many bacteria (i.e. Enterobacteriaceas, Spirochaeta,
Bacteroides, etc.) metabolize glucose by the
Embden-Meyerhof pathway. Briefly, this path utilizes
1 mol of glucose to yield 2 mol of pyruvate which are
Am. J. Sci. Ind. Res., 2011, 2(4): 694-706
glucose and other simple sugars destroys much of
the sugars in the process.
One way of making cellulose wastes more
susceptible to hydrolysis is by subjecting them to a
short burst of high energy electron beam radiation.
An alternative to acid hydrolysis is the use of
enzymes.
Although they avoid the corrosion problems and loss
of fuel product associated with acid hydrolysis,
enzymes have their own drawbacks. Enzymatic
hydrolysis slows as the glucose product accumulates
in a reaction vessel. This end-product inhibition
eventually halts the hydrolysis unless some way is
found to draw off the glucose as it is formed. (12)
Ethanol Fermentation with Bacteria: A great
number of bacteria are capable of ethanol formation.
Many of these microorganisms, however, generate
multiple end products in addition to ethyl alcohol.
These include other alcohols (butanol,
isopropylalcohol, 2,3-butanediol), organic acid (acetic
acid, formic acid, and lactic acids), polyols (arabitol,
glycerol and xylitol), ketones (acetone) or various
gases (methane, carbon dioxide, hydrogen).(12)
Those microbes that are capable of yielding ethanol
as the major product (i.e. a minimum of 1 mol ethanol
produced per mol of glucose utilized) are shown in
the table below.
mmol Ethanol Produced per Mmol Glucose Metabolized
up to 4.15 a)
1.96 a)
a)
1.8 (1.8) b)
1.7
1.9
1.7
1.5 (0.8)
0.84 (1.46)
1.1 (1.4)
1.2
1.1
1.0
1.0
then de-carbosylated to acetaldehyde and reduced to
ethanol. (12) Although many Bacteria metabolize
glucose by the Embden-Meyerhof pathway, the
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fermentation can also be analyzed using Michaelis - magnesium must also be provided for the synthesis
Menten model for certain bacteria (2) of minor components. Minerals (i.e. Mn, Co, Cu, Zn)
and organic factors (amino acids, nucleic acids, and
A close look at Zymomonas Mobilis as an ethanol vitamins) are required in trace amounts.
Producing Bacteria
Zymomonas mobilis Yeast are highly susceptible to ethanol inhibition.
Concentrations of 1-2% (w/v) are sufficient to retard
microbial growth and at 10% (w/v) alcohol, the growth
Scientific Classification
rate of the organism is nearly halted.
Kingdom: Bacteria
Saccharomyces cerevisiae
Phylum: Proteobacteria
Scientific classification
Class: Alpha Proteobacteria
Kingdom: Fungi
Order: Sphingomonadales
Phylum: Ascomycota
Family: Sphingomonadaceae
Subphylum: Saccharomycotina
Genus: Zymononas
Class: Saccharomycetes
Species: Z. mobilis
Order: Saccharomycetales
Family: Saccharomycetaceae
Zymomonas mobilis is a bacterium belonging to the
Genus: Saccharomyces
genus Zymomonas. It is notable for its bioethanol-
Species: S. cerevisiae
producing capabilities, which surpass yeast in some
Binomial name Saccharomyces cerevisiae
aspects. It was originally isolated from alcoholic
Comparison of Z. mobilis and S. cerevisiae with
beverages like the African palm wine, the Mexican
respect to producing bio-ethanol:
pulque, and also as a contaminant of cider and beer
in European countries.
ADVANTAGES OF Z. Mobilis over S. cerevisiae
Z. mobilis degrades sugars to pyruvate using the
Entner-Doudoroff pathway. The pyruvate is then i. higher sugar uptake and ethanol yield,
fermentated to produce ethanol and carbon dioxide ii. lower biomass production,
as the only products (analogous to yeast). (13) iii. higher ethanol tolerance,
Ethanol Fermentation with Yeast iv. does not require controlled addition of
The organisms of primary interest to industrial oxygen during the fermentation,
operations in fermentation of ethanol include v. Amenability to genetic manipulations.
Saccharomyces cerevisiae, Saccharomyces uvarum, Disadvantages of Z. Mobilis compared to S.
Schizosaccharomyces pombe, and Kluyveromyces cerevisiae
sp. Yeast, under anaerobic conditions; metabolize Its utilizable substrate range is restricted to glucose,
glucose to ethanol primarily by way of the Embden- fructose, and sucrose. Using biotechnological
Meyerhof pathway. The overall net reaction involves methods, scientists are currently trying to overcome
the production of 2 moles each of ethanol, but the this. A variant of Z. mobilis that is able to use certain
yield attained in practical fermentations however pentoses as a carbon source has been developed.
does not usually exceed 90 – 95% of theoretical. This An interesting characteristic of Z. mobilis is that its
is partly due to the requirement for some nutrient to plasma membrane contains hopanoids, pentacyclic
be utilized in the synthesis of new biomass and other compounds similar to eukaryotic sterols. This allows
cell maintenance related reactions. it to have an extraordinary tolerance to ethanol in its
environment, around 13%. (13)
A small concentration of oxygen must be provided to
the fermenting yeast as it is a necessary component PROCEDURE AND METHODOLOGY
in the biosynthesis of polyunsaturated fats and lipids.
Materials used in the experimentation
Typical amounts of O2 maintained in the broth are
0.05 – 0.10 mm Hg oxygen tension. • Erlenmeyer flasks
• Test tubes
The relative requirements for nutrients not utilized in
• Rubber pipe or glass tube
ethanol synthesis are in proportion to the major
• Retort stand
components of the yeast cell. These include carbon
oxygen, nitrogen and hydrogen. To lesser extent • Reflux condenser
quantities of phosphorus, sulfur, potassium, and • CaOH

697

• Stopper/Cork
• Stop watch
• Thermometer
• Molasses
• Yeast (Saccharomyces Cervisae)
• 600ml beakers
• Funnel
• Filter paper
• pH meter
PROCESS DESCRIPTION
BASIC EXPERIMENTAL PROCEDURE (3)
Pre-treatment of molasses
70 ml of molasses was mixed with 70 ml of water in a
250 ml Erlenmeyer flask.
About 0.5 g of yeast was added to the flask and stir
gently until everything is well mixed.
This reaction was stored in a drawer for one week
while the fermentation reaction occurs. A simple
distillation column was prepared; the simple
distillation was done fairly rapidly (one drop per
second) and the alcohol fraction should be collected
until just below the boiling point of water.
The ethanol was distilled slowly; recording the
temperature range for each fraction collected,
stopping collection at 97°C.
The above procedures were repeated for different
grams of yeast varying from 0.5 grams of yeast to
7grams of yeast and for each gram of yeast the
quantity of water was varied from 35ml to 280ml. The
results are recorded and discussed in the succeeding
chapter.
Precautions taken during the experimentation.
i. The molasses was refrigerated until the
experimental date to avoid unwanted action
of bacteria.
ii. The yeast was also refrigerated until the
experimental date to avoid it being activated.
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iii. The flask was stopped with a one-hole
rubber stopper containing a bent glass tube.
A short rubber hose was attached to the bent
glass tube and a short straight section of
glass tube was inserted into the other end of
the rubber tube. The straight glass tube was
dipped into a test tube two-thirds full of
limewater (Ca (OH) 2 solution). The test tube
of limewater serves as a one-way vapour
lock, keeping air from entering the flask while
allowing the carbon dioxide to escape.
The results for the experiment are recorded in the
succeeding chapter.
RESULTS AND DISCUSSION OF RESULTS
Due to the quality of the analysis especially the
distillation, a relatively good yield of ethanol is
expected, although maybe not the best.
CHARACTERISTICS OF RAW MATERIAL
Table 4.1 shows the average analytical properties of
the molasses from BUA sugar refinery.
The analysis was carried out as follows:
i. To find the Brix was used at the lab; a portion
of the molasses was poured into the sensor
of a Brixometer and the reading was taken.
ii. To find the purity 13g of the sample was
taken in a beaker and the sample was made
up with water to the dilute brix value after
which a clarifying reagent called octapol is
added and the resulting solution is filtered to
get a golden clear liquid then the solution is
poured into a saccharimeter and the pol
value is taken an the purity (amount of
sucrose per sample) is calculated (see
appendix B for calculation of purity)
iii. For the Ash and pH values a simple
conductivity meter was used to determine the
values by dipping the electrodes in the
sample.
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Table 4. 1 Characteristic Properties of the Molasses used for the analysis

Property Value
Brix 73.210
Dilute Brix 36.605
Purity 64.530
Ash 8.238%
pH 5.097
Temp 30˚C
Colour Dark consistent brown
Density 1.292

TEST EXPERIMENT RESULTS Weight of specific gravity bottle + aqueous ethanol

Table 4.2 Test Run Experiment: Premium Yeast (B) =50.58 g
Quantity For The Best Yield Of The Ethanol Weight of aqueous ethanol = B-A= 21.88g
Produced. Volume of ethanol = 50ml
Specific gravity of ethanol (ρ) = m = 0.9338g/ml
The values for the table below were gotten as V
follows: Gram ethanol present per 100ml = 42.798g
Ratio of water to molasses = 1:1 (varied for other Note: the above calculation was repeated for all
cases see Appendix II) the tabulated results henceforth.
Amount of yeast = 0.2g
Weight of specific gravity bottle (A) = 28.70g
S/N Weight of Yeast (g) Density of Sample Wt% of ethanol gram ethanol per
(g/ml) produced 100ml
1 0.200 0.915 46.750 42.798
2 0.500 0.947 31.500 29.853
3 1.200 0.968 20.000 19.373
4 3.000 0.934 40.000 37.380
5 5.000 0.885 62.270 55.108

Table 4.2 reveals that the increase in the quantity of
yeast does not favour the process initially but with the
continuous increase the yeast activity increases
thereby increasing the yield. New substances might
have been formed, but they are still held within the
mash while the gases (CO2 and possibly minute
quantities of methane) were trapped in CaOH,
Observing that 5 grams of yeast gave the maximum
ethanol yield the main experimental analysis would
be carried out around these ranges (from 1 gram to
Table 4.3: Premium yeast quantity for the best yield of ethanol analysis (1 gram of yeast)
7grams with no definitive spacing in the quantities
just random weights).
ANALYSIS OF THE EFFECT OF DILUTION RATIO
The various dilution ratios are as follows 1: 0.5; 1: 1;
1: 2; 1: 3; 1: 4 (being 1 part molasses to 1 part water,
1 part molasses to 2 parts water etc respectively)
represented as decimals on the chart, theses ratios
would be used for the yeast variations as would be
analyzed hitherto.
4.2.2.1 Analysis for samples fermented with 1 gram
of yeast

1gram DENSITY AND WT % OF THE ETHANOL

Ratio Of Molasses To Water DENSITY WT % 1gram (gram ethanol per 100ml )
0.667
0.888 57.781 52.959
0.500
0.888 57.868 53.028
0.333
0.881 60.991 55.524
0.250
0.863 68.296 61.211
0.200
0.816 86.556 74.492

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Fig 4.1: Chart of Dilution Ratio to yield after 5 days of Fermentation (1 gram of yeast)

Analysis of figure 4.1

Fig 4.2: Chart of Dilution Ratio to yield after 5 days of Fermentation (2 grams of yeast)
Analysis of figure 4.2

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According to the chart of the experimental result the

yield was best at ratio 1:0.5 (0.2 molasses
concentration). From literature this could be caused
by the insufficient sugar quantity available for
fermentation as the more diluted the culture the less
the yield meaning the yeast had very little to ferment
thereby producing less ethanol as the quantity of
water increased because as the biomass is diluted,
the sugar level gradually washes out. Note also that
the time taken for the breakdown process might have
affected the time available for the
4.2.2.2 Analysis for 2 grams of yeast.
According to the chart of the experimental result the
yield was best at ratio 1:2 (0.3333 molasses
concentration). From literature this could be caused
by the insufficient glucose concentrations at the
higher dilution ratios while at lower ratios the glucose
quantity might be too high therefore the yeast might
not act as well also several strains (e.g. K.
marxianus, S.uvarum) have been specially cultured
for this purpose (high sugar content mediums).

yeast to convert most of the converted glucose to

ethanol before being worn out (or requiring
regeneration) due to the quantity of yeast available.

Table 4. 4 Premium yeast quantity for the best yield of ethanol analysis (2 grams of yeast)

2grams DENSITY AND WT % OF THE ETHANOL

RATIO OF MOLASSES TO WATER DENSITY WT % 2grams (gram ethanol per 100ml )
0.667
0.924 40.976 38.851
0.500
0.919 43.630 41.154
0.333
0.855 71.614 63.724
0.250
0.938 33.802 32.485
0.200
0.979 10.433 10.319
4.2.2.3 Analysis for 3 grams of yeast

Table 4.5: Premium yeast quantity for the best yield of ethanol analysis (3 grams of yeast)

RATIO OF MOLASSES TO WATER DENSITY WT % for 3 grams (gram ethanol per 100ml )
0.667 0.934 36.208 34.643
0.500 0.938 34.224 32.865
0.333 0.930 37.955 36.195
0.250 0.977 11.706 11.582
0.200 0.974 13.404 13.257

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Fig 4.3: Chart of Dilution Ratio to yield after 5 days of Fermentation (3 grams of yeast)
Analysis of figure 4.3 breakdown process is rapid but not as rapid but also
as the sugar is being broken down reproduction takes
place for the yeast due to this divided attention the
Behaves just like the cultures with 2 grams of yeast yeast are unstable and the yield unstable in return.
only the medium is more unstable since the yeast are
not adequate to balance the culture and get a steady 4.2.2.4 Analysis for 5 grams of yeast.
graph the yeast are many therefore the sugar

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Table 4.5: Premium yeast quantity for the best yield of ethanol analysis (5 grams of yeast)

RATIO OF MOLASSES TO WATER DENSITY WT % 5grams (gram ethanol per 100ml )

0.667
0.928 39.274 37.359
0.500
0.897 53.868 49.775
0.333
0.908 48.718 45.491
0.250
0.933 36.673 35.057
0.200
0.961 21.392 20.986

Fig 4.4: Chart of Dilution Ratio to yield after 5 days of Fermentation (5 grams of yeast)
Analysis of figure 4.4
Due to the increase in yeast quantity the ethanol yield purest ethanol quality, it can also be seen that the 7
is higher within the less diluted mediums causing the grams gave the next highest this is to say that the
yield to be higher although the yield is higher than the ethanol yield reduces with an increase in yeast
previous two at the lower dilution rate regions the concentration until a critical point after which it starts
optimum yield still lays at the ratio 1:2. to increase again.
For every dilution rate the yeast behaved differently
although averagely the 1 gram of yeast gave the

4.2.2.4 Analysis for 7 grams of yeast

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Table 4. 6: Premium yeast quantity for the best yield of ethanol analysis (7 grams of yeast)

RATIO OF MOLASSES TO WATER DENSITY WT % 7grams(gram ethanol per 100ml)

0.667
0.908 48.951 45.687
0.500
0.925 40.478 38.416
0.333
0.912 46.841 43.903
0.250
0.829 81.483 70.936
0.200
0.960 21.802 21.375

Verification Analysis of sample using a UV visible Spectrometer

This verification analysis is based strictly on the comparison of the absorbance of each substance with a pure

stock sample.
Figure A.UV Spectroscopy Test result

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Fig A.UV Spectroscopy Test result 2

Comparing the two samples based on their wavelengths and absorbance it can be deduced that the samples
are relatively close in their electronic transitional structure since they both have an absorbance of close range
and this occurs at the same wavelength.

Fig 5. 1: Combined Chart for the Ethanol Yield

CONCLUSION chart shows that yield is highest at more dilution

From the chart above it would be observed that after
the 1 gram yeast concentration the 5 gram yeast
concentration gave the highest yield, this validates
the region chosen for analysis (between 1gram and
5grams of yeast). Although this experimental data
taking 1 gram of yeast into consideration as this
seems to have a more systematically defined pattern.
It can be concluded that the yield of ethanol is greatly
dependent on the quantity of fermentable sugars
present in the biomass.

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