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: Ethanol, also called ethyl alcohol, pure alcohol, grain
The aim of this project is to study the dilution rate of alcohol, or drinking alcohol, is a volatile, flammable,
molasses and the effect of yeast on the yield of colourless liquid. In common usage, it is often
ethanol using batch fermentation. referred to simply as alcohol or spirits. Ethanol is a
straight-chain alcohol, and its molecular formula is
METHOD AND SCOPE OF STUDY C2H5OH. Its empirical formula is C2H6O. An
alternative notation is CH3-CH2-OH, which indicates
Various methods can be used for the specific
that the carbon of a methyl group (CH3-) is attached
purpose of the production of ethanol; this includes
to the carbon of a methylene group (-CH2-), which is
Ethylene hydration, Fermentation, Cellulosic ethanol.
attached to the oxygen of a hydroxyl group (-OH). It
The above stated methods are pathways to ethanol is a constitutional isomer of di-methyl ether. Ethanol
production but fermentation is applied in this paper. is often abbreviated as EtOH, using the common
organic chemistry notation of representing the ethyl
RELEVANCE OF STUDY group (C2H5) with Et.
This study poses great opportunities for the chemical Ethanol burning with its spectrum depicted. (5)
engineering profession in Covenant University and Hydrogen bonding causes pure ethanol to be
Nigeria at large. hygroscopic to the extent that it readily absorbs water
from the air. The polar nature of the hydroxyl group
 Am. J. Sci. Ind. Res., 2011, 2(4): 694-706

causes ethanol to dissolve many ionic compounds, Finally, one of the hydrogens on the oxygen is
notably sodium and potassium hydroxides, removed by reaction with the dihydrogenphosphate
magnesium chloride, calcium chloride, ammonium (V) ion, H2PO4-, formed in the first step.
chloride, ammonium bromide, and sodium bromide.(4)
Sodium and potassium chlorides are slightly soluble The reversibility of this reaction (7)
in ethanol.(4) Because the ethanol molecule also has This reaction is entirely reversible - and so each step
a non polar end, it will also dissolve non-polar is reversible. In the reverse direction, this is a
substances, including most essential oils (6) and dehydration of ethanol to make ethene.
numerous flavoring, coloring, and medicinal agents. Equations of reaction
The addition of even a few percent of ethanol to C2H4 (g) + H2O (g) → CH3CH2OH (l).-------------Equation
water sharply reduces the surface tension of water. 2.1
This property partially explains the “tears of wine” C2H4 + H2PO4 → CH3CH2PO4H ---------------Equation
phenomenon. Mixtures of ethanol and water that 2.2
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 Fig 2 . 2: Ethylene hydration using phosphoric acid
catalyst
of the ethanol is exothermic.
CH3CH2PO4H + H2O → CH3CH2OH + H2PO4-----------
Only 5% of the ethylene is converted into ethanol at Equation 2.3
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 Fig 2.3: Phosphoric acid catalyst regeneration
All of the hydrogen atoms in the phosphoric (V) acid Fermentation
are fairly positively charged because they are
The breakdown of carbohydrates to ethanol, carbon
attached to a very electronegative oxygen atom.
di-oxide and water using micro organisms. (10)
One of these hydrogen’s is strongly attracted to the The many and varied raw materials used in the
carbon-carbon double bond. manufacture of ethanol via fermentation are
conveniently classified under three types of
The pi part of the bond breaks and the electrons in it
agricultural raw materials (11):
move down to make a new bond with the hydrogen
1. Sugar
atom. That forces the electrons in the hydrogen-
2. Starches
oxygen bond down entirely onto the oxygen.
3. Cellulose materials.
Step 2 Fermentation from Sugars: Sugars from sugar
cane, sugar beets, molasses, and fruits) can be
The carbocation (carbonium ion) formed reacts with converted to ethanol directly.
one of the lone pairs on a water molecule. A
carbocation is one which carries a positive charge on The most widely used sugar for ethanol fermentation
a carbon atom. (7) is blackstrap molasses which contains about 35 – 40
wt% sucrose, 15 – 20 wt% invert sugars such as
Step 3 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

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to a mash containing 10 –20 wt% sugar. After the pH glucose and other simple sugars destroys much of
of the mash is adjusted to about 4 – 5 with mineral the sugars in the process.
acid, it is inoculated with the yeast, and the
fermentation is carried out non-aseptically at 20 – One way of making cellulose wastes more
32°C for about 1 – 3days. The fermented beer, which susceptible to hydrolysis is by subjecting them to a
typically contains 6 – 10 wt% ethanol, is then set to short burst of high energy electron beam radiation.
the product recovery in purification section of the An alternative to acid hydrolysis is the use of
plant. enzymes.
Fermentation from Starches: All potable alcohol Although they avoid the corrosion problems and loss
and most fermentation industrial alcohol are currently of fuel product associated with acid hydrolysis,
made principally from grains. Fermentation of starch enzymes have their own drawbacks. Enzymatic
from grain is somewhat more complex than hydrolysis slows as the glucose product accumulates
fermentation of sugars because starch must first be in a reaction vessel. This end-product inhibition
converted to sugar and then to ethanol (12). Starch is eventually halts the hydrolysis unless some way is
converted enzymatically to glucose either by diastase found to draw off the glucose as it is formed. (12)
presents in sprouting grain or by fungal amylase. The Ethanol Fermentation with Bacteria: A great
resulting dextrose is fermented to ethanol with the aid number of bacteria are capable of ethanol formation.
of yeast producing CO2 as co-product. A second co- Many of these microorganisms, however, generate
product of unfermented starch, fiber, protein and ash multiple end products in addition to ethyl alcohol.
known as distillers grain (a high protein cattle feed) is These include other alcohols (butanol,
also produced. isopropylalcohol, 2,3-butanediol), organic acid (acetic
Fermentation from Cellulosic Materials: Each step acid, formic acid, and lactic acids), polyols (arabitol,
in the process of the conversion of cellulose to glycerol and xylitol), ketones (acetone) or various
ethanol proceeded with 100% yield; almost two-thirds gases (methane, carbon dioxide, hydrogen).(12)
of the mass would disappear during the sequence,
most of it as carbon dioxide in the fermentation of Those microbes that are capable of yielding ethanol
glucose to ethanol. This amount of carbon dioxide as the major product (i.e. a minimum of 1 mol ethanol
leads to a disposal problem rather than to a raw produced per mol of glucose utilized) are shown in
material credit. Another problem is that the aqueous the table below.
acid used to hydrolyze the cellulose in wood to
Table 2. 1 : Bacteria useful for fermentation
Mesophilic Organisms mmol Ethanol Produced per Mmol Glucose Metabolized

Clostridium sporogenes up to 4.15 a)

Clostridium indolis 1.96 a)
(pathogenic)
a)
Clostridium sphenoides 1.8 (1.8) b)
Clostridium sordelli 1.7
(pathogenic)
Zymomonas mobilis 1.9
(syn.Anaerobica) (anaerobe)
Zymomonas mobilis 1.7
Ssp. Pomaceas
Spirochaeta aurantia 1.5 (0.8)
Spirochaeta stenostrepta 0.84 (1.46)
Spirochaeta litoralis 1.1 (1.4)
Erwinia amylovora 1.2
Leuconostoc mesenteroides 1.1
Streptococcus lactis 1.0
Sarcina ventriculi 1.0
(syn. Zymosarcina)
Many bacteria (i.e. Enterobacteriaceas, Spirochaeta, then de-carbosylated to acetaldehyde and reduced to
Bacteroides, etc.) metabolize glucose by the ethanol. (12) Although many Bacteria metabolize
Embden-Meyerhof pathway. Briefly, this path utilizes glucose by the Embden-Meyerhof pathway, the
1 mol of glucose to yield 2 mol of pyruvate which are

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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

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• Stopper/Cork iii. The flask was stopped with a one-hole

• Stop watch rubber stopper containing a bent glass tube.
• Thermometer A short rubber hose was attached to the bent
• Molasses glass tube and a short straight section of
• Yeast (Saccharomyces Cervisae) glass tube was inserted into the other end of
• 600ml beakers the rubber tube. The straight glass tube was
• Funnel dipped into a test tube two-thirds full of
limewater (Ca (OH) 2 solution). The test tube
• Filter paper
of limewater serves as a one-way vapour
• pH meter
lock, keeping air from entering the flask while
allowing the carbon dioxide to escape.
PROCESS DESCRIPTION
The results for the experiment are recorded in the
BASIC EXPERIMENTAL PROCEDURE (3)
succeeding chapter.
Pre-treatment of molasses
70 ml of molasses was mixed with 70 ml of water in a RESULTS AND DISCUSSION OF RESULTS
250 ml Erlenmeyer flask.
About 0.5 g of yeast was added to the flask and stir Due to the quality of the analysis especially the
gently until everything is well mixed. distillation, a relatively good yield of ethanol is
This reaction was stored in a drawer for one week expected, although maybe not the best.
while the fermentation reaction occurs. A simple CHARACTERISTICS OF RAW MATERIAL
distillation column was prepared; the simple
distillation was done fairly rapidly (one drop per Table 4.1 shows the average analytical properties of
second) and the alcohol fraction should be collected the molasses from BUA sugar refinery.
until just below the boiling point of water. The analysis was carried out as follows:
The ethanol was distilled slowly; recording the i. To find the Brix was used at the lab; a portion
temperature range for each fraction collected, of the molasses was poured into the sensor
stopping collection at 97°C. of a Brixometer and the reading was taken.
The above procedures were repeated for different ii. To find the purity 13g of the sample was
grams of yeast varying from 0.5 grams of yeast to taken in a beaker and the sample was made
7grams of yeast and for each gram of yeast the up with water to the dilute brix value after
quantity of water was varied from 35ml to 280ml. The which a clarifying reagent called octapol is
results are recorded and discussed in the succeeding added and the resulting solution is filtered to
chapter. get a golden clear liquid then the solution is
poured into a saccharimeter and the pol
Precautions taken during the experimentation. value is taken an the purity (amount of
sucrose per sample) is calculated (see
i. The molasses was refrigerated until the
appendix B for calculation of purity)
experimental date to avoid unwanted action
iii. For the Ash and pH values a simple
of bacteria.
conductivity meter was used to determine the
ii. The yeast was also refrigerated until the
values by dipping the electrodes in the
experimental date to avoid it being activated.
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 7grams with no definitive spacing in the quantities
yeast does not favour the process initially but with the just random weights).
continuous increase the yeast activity increases ANALYSIS OF THE EFFECT OF DILUTION RATIO
thereby increasing the yield. New substances might The various dilution ratios are as follows 1: 0.5; 1: 1;
have been formed, but they are still held within the 1: 2; 1: 3; 1: 4 (being 1 part molasses to 1 part water,
mash while the gases (CO2 and possibly minute 1 part molasses to 2 parts water etc respectively)
quantities of methane) were trapped in CaOH, represented as decimals on the chart, theses ratios
Observing that 5 grams of yeast gave the maximum would be used for the yeast variations as would be
ethanol yield the main experimental analysis would analyzed hitherto.
be carried out around these ranges (from 1 gram to 4.2.2.1 Analysis for samples fermented with 1 gram
of yeast
Table 4.3: Premium yeast quantity for the best yield of ethanol analysis (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 4.2.2.2 Analysis for 2 grams of yeast.
concentration). From literature this could be caused According to the chart of the experimental result the
by the insufficient sugar quantity available for yield was best at ratio 1:2 (0.3333 molasses
fermentation as the more diluted the culture the less concentration). From literature this could be caused
the yield meaning the yeast had very little to ferment by the insufficient glucose concentrations at the
thereby producing less ethanol as the quantity of higher dilution ratios while at lower ratios the glucose
water increased because as the biomass is diluted, quantity might be too high therefore the yeast might
the sugar level gradually washes out. Note also that not act as well also several strains (e.g. K.
the time taken for the breakdown process might have marxianus, S.uvarum) have been specially cultured
affected the time available for the 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

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

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