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Bioethanol production from sugar cane molasses by using

Zymomonas Mobilis
Asif Hussain1 , Abeera A. Ansari1 , Azra Nawar1 , Hamza Ehsan2 and Ehsan Ali1
Centre for Energy Systems, National University of Science and Technology Islamabad, (Pakistan)
Atta Ur Rehman School of Applied & Biosciences, National university of Science and Technology Islamabad, (Pakistan)

A BSTRACT years [7]. The utilization of sugar cane molasses for

The strain Zymomonas Mobilis was used to produce fermentation process is one of the oldest chemical
bioethanol from sugar cane molasses. The study was process known to human. This practice is also widely
conducted to investigate the optimized conditions for adopted for bioethanol production[1][2]. In recent years,
production of bioethanol through batch fermentation. The however many of the products are synthesized chemically
fermentation unit was designed to determine the effect of from petroleum feedstock including bioethanol often cost
fermentation temperature, pH, sugar concentration and effectively. It depends upon the specificity of the
supply of nutrients. The Zymomonas Mobilis produced microorganism and to get the useful products like
9.3% (v/v) bioethanol by utilizing 16gm/100ml sugar bioethanol [8]. From last three decades , studies were
with the fermentation efficiency 90.5%. The nutrients carried out to minimize the issue in the fermentation
have been supplied to enhance the production of technology for efficient bioethanol production [9][10].
fermentation yield. The pH and fermentation temperature The yeast (Saccharomyces cerevisiae) has been used
were optimized for the improved yield of bioethanol widely in commercial scale but bacteria (Zymomonas
mobilis) has not been commercially practiced yet due to
Keywords: Batch Fermentation, Bioethanol, Molasses, some constraints. Bacteria has some advantages over
Zymomonas Mobilis yeast as studied by Sadik et al [11] [12]. Yeast has its
own limitations as compared to bacteria. Different studies
INT RODUCT ION were conducted to sort out the concerned issue in bacteria
fermentation [13][14].In this study, the factors effecting
The depletion of fossil fuels has caused a significant
in the bioethanol production were investigated. The study
increase in the oil prices since last two decades[1].
was developed for the optimum yield of bioethanol by
Pakistan imports 6.939 million tons of oil for the year
using Z. mobilis in prime conditions.
2012-13 to meet its energy requirements [2]. With
reference to current energy scenario, Pakistan needs to
pay special attention on alternative fuels, as the feedstock
is widely available. Among the biofuels, bioethanol is
quiet notable and widely produced as an important fuel A. Sugar Cane Molasses
worldwide [1]. The literature records that the bioethanol Sugar cane molasses was provided by Noon Sugar
can be used in substitution of gasoline in existing engines mills Pvt. Limited Bhulwal Pakistan.
or it can be blended, producing low emissions of
B. Strain
greenhouse gases (GHG)[3]. Bioethanol can be produced
by utilizing the biomass, molasses, or any lignocellulosic The strain Zymomonas Mobilis was used in this study.
material with the help of microorganisms. The feedstock Z. mobilis (DSM 424) was procured from DSMZ1
used in this study is molasses, which is widely available Germany.
in sugar producing regions [4]. Pakistan is producing 2- C. Bacteria (Zymomonas Mobilis) Medium
2.5 million tons of molasses every year and 80%
Zymomonas media suggested by DSMZ Germany,
molasses are being exported annually [5]. If this product
bacto-peptone 10 gm/L, yeast extract 10 g/L, 15 gm/L
is utilized locally for the production of bioethanol, it may
agar for agar plates, glucose 20gm/L, distilled water mark
be able to contribute 2-3% in transportation fuel
up to 1000ml and with pH 7.00[15]. Media was
production annually [6].
autoclaved at 121C for 45 minutes , streaking was done
Molasses is a byproduct of sugar industry having on agar plates in Desktop Laminar Flow, and was placed
significant quantity of sugar, around 40% to 50%(w/v), in incubator (Syno Japan) for overnight.. The growth
and ash content around 5-15%, making it a suitable cells were analyzed by Hemocytometer and were
substrate for rum and bioethanol production since many

Leibniz-Institut DSMZ-Deutsche Sammlung von
Mikroorganismen und Zellkulturen GmbH

3 rd International Conference on Energy, Environment & Sustainable Development (EESD2014)

MUET Jamshoro, Pakistan, October 22-24, 2014
inoculated into test tubes for scaling up and also stored in determined on the basis of volatility. The boiling point of
glycerol stock for preservation. water, used in the process was set as reference
temperature. The pH has been determined by pH meter
D. Preparation of Molasses medium
handy (EZDO 6011 Japan)
The 250 ml molasses was taken in a conical flask for
I. Acidity Test
pre-fermentation with the sugar concentration of
7gm/100L inoculated with 5ml growth media. The cells 10 ml fermented sample was taken in beaker and put
were counted. The desirable cell growth was achieved in the stirrer and pH meter dipped in this sample to check
and used for fermentation. the pH. The sample was taken in burette, titrated the
sample by 1N NaOH solution till its pH reached to 7.00.
E. Batch Fermentation The reading was noted and multiplied by 0.69, which is
The known amount of sugar cane molasses and grown equivalent weight of sulphuric acid. The acidity values
media were taken in fermentation flask and kept in shaker were calculated by this method.
for fermentation. An anaerobic condition was maintained
J. Fermentation efficiency
and kept for two days, the strains converted sugar into
bioethanol with the evolution of CO2 . The samples were The fermentation efficiency was calculated by the
tested after 48 hours, to investigate the effect of sugar given formula.
concentration, pH, fermentation temperature and supply F.E = (actual yield / theoretical yield) 100
of nutrients. Where F.E = fermentation efficiency.

F. Detection of Bioethanol RESULT & DISCUSSIONS

5 mL of fermented sample was taken and pinch of A. Effect of pH
potassium dichromate and few drops of Concentrated
H2 SO4 were added. The brownish color of sample was The samples were fermented in different pH ranges
changed into green that indicated the presence of from 4.0 to 6.0 to obtain the maximum yield of
bioethanol. bioethanol by adding diluted sulphuric acid. The sugar
concentration and temperature were kept constant.
G. Analysis for total sugar concentration in Molasses Anaerobic conditions were applied and the fermented
The sugar concentration in molasses was determined samples were analyzed after 48 hours [19]. Table I show
through Fehlings test[16][17]. The 5ml molasses sample the bioethanol yields and fermentation efficiency.
was taken dissolved in 100ml of distilled and 5ml of T ABLE I
concentrated Hcl and was heated at 70C for 10 minutes EFFECT OF P H ON FERMENT AT ION T HROUGH Z.MOBILIS
and then it was neutralized by adding NaOH. It was (ZM 424)
prepared to 1000ml and then taken into burette solution. Sugar
F. EtoH5 yield
pH Conc. Acidity F.E%
The 5mL of Fehling A 2 and Fehling B3 solution were temp4 (v/v)%
taken and mixed with 10 ml of distilled water in a conical
4 14 33 8.1 4.4 49
flask and methylene blue indicator was added. The
solution was titrated in boiling conditions until the blue 4.2 14 33 6.3 6.5 72
color disappeared. The sugar concentration was 4.4 14 33 5.2 6.8 75
calculated by following formula: 4.6 14 33 4.9 7.1 79

4.8 14 33 4.2 7.7 85
TS =
TV 100 5 14 33 4 7.9 88
5.2 14 33 6.5 6.2 69
Where TS = Total sugars, DF= Dilution factor 5.4 14 33 7.5 4.7 52
5.6 14 33 6.3 2.2 24
FF=Fehling factor, TV=Titrate value
5.8 14 33 13.8 0.0 0
The second method to find the sugar concentration is the
use of portable Refractometer RHB 32ATC Japan, it has The maximum yield 7.9 (v/v) was achieved on pH 5.0
limitations, and it can measure the maximum and fermentation efficiency was recorded 88%showed in
concentration of sugar up to 16 gm/100mL. Total sugar Table I. While lowering the pH showed less yield and
found in used molasses was 47%. production of acids, which was confirmed by acidity test.
H. Determination of bioethanol concentration and pH pH higher than 5.4 revealed a smaller amount of/or no
yield. Yanase et al also observed the maximum
The different bioethanol concentrations were
productivity at 4.8 t 5.4 pH [20][7]. Z. Mobilis can
determined by Ebulliometer (J. SALLERON DUJARDIN
produce optimal yield in the high pH. The elevated
Sr. PARIS) which is approved in distilleries [18] and it
tolerance towards pH depicts that bacteria will consume
fewer sulphuric acid to maintain the pH in fermentation.
Fehling's "A" contains 7 g CuSO4.5H2 O dissolved in distilled water
containing 2 drops of dilute sulfuric acid.
3 4
Fehling's "B" contains 35g of potassium tartrate and 12g of NaOH in F.temp= fermentation temperature
100 ml of distilled water. EtOH= Ethanol

3 rd International Conference on Energy, Environment & Sustainable Development (EESD2014)

MUET Jamshoro, Pakistan, October 22-24, 2014
As we know that the molasses pH is range in between 5.0 T ABLE III
to 6.0. It is a major challenge to maintain pH in the
fermentation. This study also reveals that the optimum
pH for Z.mobilis is in the range of 4.0 t0 4.6 [21] Sugar Density
EtoH F.
Conc. of PH T emp Acidity
[22][23]. yield E
(gm/100ml) Molasses
B. Effect of fermentation temperature
The samples were maintained in the optimum pH 10 1.079 5 33 6.2 4.70% 73
5.0 for Z.mobilis with the fermentation temperature 12 1.097 5 33 5.9 6.20% 80.4
ranging from 28C, 30C, 32C, 34C, 36C and 38C
[Table II]. 14 1.116 5 33 5.2 7.80% 86.7

T ABLE II 16 1.135 5 33 4.4 8.9% 87

FERMENT AT ION BY USING Z.MOBILIS (ZM 424) 18 1.155 5 33 9.4 5.5% 50

F. T emp Sugar Conc. F.E Above analysis indicated that the Z.mobilis has capability
pH Acidity yield
C (gm/100ml) %
(v/v)% to tolerate the higher sugar content with better efficiency.
Cazetta et al (2007) studied the sugar concentration effect
28 5 14 7.4 3.1 34.5 20gm/100ml but the enzymes were used for optimum
30 5 14 5.2 5.2 58 yield. Due to enzymes the Z.mobilis tolerated higher
sugar concentrations [25].
32 5 14 4.4 7.6 84.5
D. Effect of nutrients
34 5 14 4.1 7.9 87.6
Fadel et al (2013) studied the effect of different
36 5 14 6.7 5.8 64.5 nutrients in an anaerobic fermentation. Nutrients are quite
38 5 14 8.1 2.2 25
effective in the production of bioethanol from sugar cane
molasses [26]. Di-Ammonium phosphate (DAP) and
Urea were supplied to the fermentation with pre-optimum
The samples were kept for fermentation for 48 hours, parameters. The contribution of the DAP and urea in the
after analyzing the samples it was found that the optimum process were 1:1. Different nutrients can be supplied for
yield was achieved at 34C. At that temperature the fermentation process, the most effective nutrients
bioethanol yield was 8%(v/v) along with the fermentation found are DAP and Urea reported by Fadel et al (2013).
efficiency of 88.96% showed in Table II. The increase in But their quantity has been adjusted according to our set
temperature increased bioethanol yield till 34C,
furthermore, the yield decreased by increasing T ABLE IV
fermentation temperature. The effect of temperature for EFFECT OF NUT RIENT S IN T HE FERMENT AT ION FOR
bacterial fermentation was studied by Doelle & Doelle F. EtoH
(1990) and Panesar et al (2007), both recorded 35 C as Nutrients Sugar Conc. F.E
PH T emp Acidity yield
optimum temperature [24][7]. (g/L) (gm/100mL) %
C (v/v)%
1 5 16 33 4.4 8.9 86.6
Z.mobilis produced the maximum yield at high 2 5 16 33 4.1 9.3 90.5
temperature, it was observed that it can be used in a high
3 5 16 33 4.2 9.1 88.6
temperature process. The effect of fermentation
temperature were studied by Cazetta et al (2007) and
found 32C as optimum for Z.Mobilis [25]. The values The yield increases with the addition of nutrients in the
may be quite varying because during fermentation the fermentation process with a notable efficiency.
temperature increases due to exothermic reaction.
C. Effect of Sugar molasses concentration Table IV shows that the 2 gm /L dose was found as best
quantity to get the optimum yield of 9.30%(v/v) with the
Sugar molasses concentration was set from fermentation efficiency 90.5%. The addition of nutrients
10gm/100mL to 18gm/100ml by keeping the pH and
was effective for higher sugar concentrations, no more
temperature constant (pre-optimized). Increasing sugar
effective in low concentration of sugar.
concentration the bioethanol productivity enhanced and
optimum at 16gm/100mL with 8.9 %( v/v) with the
fermentation efficiency of 87%, after this increase in CONCLUSION
concentration yielded lesser quantity of the product Table The effect of different parameters in bioethanol
III. production were studied for Z.mobilis. The optimal
condition was tabulated as 9.3%(v/v) bioethanol has
been produced with efficiency of 90.5%, supplied sugar
conc. 16gm/100mL, pH 5.0 and fermentation temperature
was found 33C. 2 gm nutrients were supplied for this

3 rd International Conference on Energy, Environment & Sustainable Development (EESD2014)

MUET Jamshoro, Pakistan, October 22-24, 2014
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MUET Jamshoro, Pakistan, October 22-24, 2014