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BIO-ETHANOL PRODUCTION FROM SUGAR CANE BY PRODUCT WITH

CHEAPEST STRAIN

Article · January 2012

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 Malaysian International Conference on Trends in Bioprocess Engineering (MICOTriBE) 2012

BE-501: BIO-ETHANOL PRODUCTION FROM SUGAR CANE BY

PRODUCT WITH CHEAPEST STRAIN

Nassereldeen. A. K, Md. Z.Alam and Sharifah Farah Syed Mokhtar

Bioenvironmental Engineering Research Centre (BERC), Biotechnology Engineering Department, Faculty of
Engineering, International Islamic University Malaysia, Gombak, 50728 Kuala Lumpur, Malaysia.
E-mail: nasreldin@iium.edu.my

Abstract
S.cerevisae is the cheapest strain available for the conversion of biomass substrate
where it is also capable to utilize variety of substrates. Sugar cane molasses contain
1688 g/l total sugars and it is chosen for its low cost and its effect of having high
yield of ethanol. The optimization of process conditions was done with different
ranges of temperature, pH, and agitation speed with fixed media compositions
obtained from previous study. It was carried out by using 2 Level Factorial design
formulated by Design Expert 6.0.8. Fermentation process was done in a BioSys 30
litres bioreactor for 24 hours. Product was analyzed for every 6 hours interval. 2
Level Factorial design was selected to design the process in order to minimize
numbers of run in bioreactor. Product analysis covers the concentration of ethanol
produced and concentration of remaining sugar. Optimum conditions for highest bio-
ethanol production using molasses are at 35 °C, pH 4.5, and 125rpm. Under this
optimum operating condition the maximum of 9.31% of ethanol was produced and
34.77% of sugars were converted into ethanol. The production is acceptable
theoretically as maximum ethanol production by wild type industrial yeasts can only
achieves maximum up 10% v/v since greater amount of ethanol produced in the
system will halt the growth of S.cerevisae. The data was validated and it was proven
that the model is well fits and the findings are reliable with R2 = 99.95%. The
objectives of the study were successfully achieved where the process parameters
were optimized and validated.

Key word: S.Cerevisae, Bioethanol, Optimization, Bioreactor

INTRODUCTION

Dependence on petroleum remains the most important factor affecting worldwide distribution of
wealth, global conflicts, and the quality of the environment. According to ([1], [2] and [3]) the research
and development of renewable energy feedstock to substitute for or to complement fossil fuel sources
have intensified by population growth and the associated demand for fuel and goods coupled with more
restrictive environmental regulations.
In attempt to maximize by-products into useful materials, molasses are used for ethanol production.
Molasses are abundance, cheap, and environmental friendly. Referring to ([4]; [5] and [6]) a good
production of bio-ethanol depends on the availability to produce high ethanol concentrations and
upholding high quality yield at the end of the process. Temperature, pH, and agitation speed are important
parameters for bio-ethanol production as they have their own role and function in order to reinsure a good

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quality eco-friendly bio-ethanol production. Optimum parameters for bio-ethanol production from
molasses are essential to minimize capital cost and to achieve a good yield of ethanol [7].
Besides, due to the increasing costs of substrates such as sweet sorghum, sugarcane, sugar beet, and
wheat starch which are human food, there is a need to search for cheaper and abundant substrate and to
develop an efficient and less expensive technology so that product can be made available more cheaply.
Leading this revolution is bio-ethanol which is made of natural, replaceable resources ([8], [9]). However,
optimizing the potential of bio-ethanol continues to be a challenge. This project brings some importance
since it responsible for bio-ethanol based on molasses production, development, and testing face highly
regulated environments and complex testing parameters. Three parameters such as pH, temperature, and
agitation speed are being optimized to achieve economic, high quality, and concentrated bio-ethanol.
Thus, optimized parameters are validating to establish optimized parameters values must be supported.
Therefore, this study may exhibit a lot of potential to use sugar cane molasses for bio-ethanol production
under validated, optimized parameters for the future use.

METHOD DEVELOPMENT

The optimization process for bio-ethanol production from molasses is the most important part in
this project. The fermentation process was done by S.cerevisae in 30 litres bioreactor. There were three
parameters involved in the optimization process, temperature, pH, and agitation speed. Aeration rate was
set to constant; 1 LPM. The optimization process was designed using Two-Level Factorial Design-Expert
6.0.8. In order to analyze and validate the product produced, several steps of upstream processes were
accomplished. Molasses were collected from Liqueur Agency Sdn Bhd, Malaysia. The concentration of
sugars in molasses was determined by phenol-sulphuric acid method. Dried-S.cerevisae obtained from
Department of Biotechnology Engineering; IIUM laboratory was inoculated in the warmed, distilled
water in order to activate the yeast. It proceeds with the downstream process, fermentation where all
parameters were set according to bioreactor experimental design. The process was done in 30 l bioreactor;
parameters were controlled and observed. Bio-ethanol was produced at the end of the process; a product
was analyzed in order to determine ethanol concentration using gas chromatography mass
spectrophotometer (GCMS). Statistical analysis was done by Analysis of Variance (ANOVA) and Design
Expert in order to validate the finding.
Flowchart shown in Figure 1 summarize an overall process for this project; optimization process
for bio-ethanol production from molasses.

Raw Material

Sugar cane molasses was a substrate for the fermentation process in this project. It was obtained
from Liqueur Agency Sdn Bhd; a molasses trader from Malaysia. It was stored at room temperature for
further usage.

Microorganism: Yeast Strain

Bio-ethanol production process in this particular project compromised cultivating Saccharomyces

cerevisae. Dried-form industrial S.cerevisae was used in this project. It was obtained from laboratory
stock. This strain is feasible for ethanol production since it shows its feasibility in previous study,
“Comparison of Sweet Sorghum and Cassava for Ethanol Production by Using Saccharomyces cerevisae”
[10].

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 Malaysian International Conference on Trends in Bioprocess Engineering (MICOTriBE) 2012

Substrate collection:
Molasses from Malaysia

Total Sugar Analysis:

Phenol-Sulphuric Acid method

Inoculums preparation: 150 Reactor preparation Media preparation

rpm, 40°C, 15 min

Acid/Base/Anti-foam Needles & tubes

preparation sterilization

Fermentation by 30 L bioreactor:
Treatment - pH, temperature, and agitation speed
Response: Bio-ethanol concentration

Production Analysis:
1. Bio-ethanol Estimation
2. Total Sugar Analysis
3. Percentage of Conversion

Production and Validation:

Bioethanol under optimized conditions

Figure 1: The flowchart of bio-ethanol production from sugar cane molasses

Standard Curve Construction

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 Malaysian International Conference on Trends in Bioprocess Engineering (MICOTriBE) 2012

Sucrose standard curve was constructed before determination of sugar in molasses was done.
Phenol-Sulphuric acid method was used for the sugar analysis. 1 ml of each sucrose sample solution with
varies concentration was dispensed in 10 ml of falcon tubes. 1 ml of 5% phenol was added in tubes
contained sucrose followed by 5 ml of 98% sulphuric acid. Solution was vortex shortly for good mixing.

Figure 2: Total Sugar Standard Curve for Sucrose

Stock Media Preparation

Theoretically, media with higher sugars content will produce higher ethanol since 40% – 50%
(w/v) of sugars will be converted into ethanol by yeasts. But inhibition of yeasts growth might be
occurring when the sugars exceed 20% (w/v). Hence, it will affect the production of ethanol. In this study,
14% - 15% of initial sugars were prepared for fermentation.
1 litre of stock media was prepared by added 60 gram of urea (0.5% w/w) and 6 gram of NPK
(0.05% w/w) with 1 l of distilled water in the Scoot bottle. The mixture was then stirred thoroughly with
magnetic stirrer for homogenize solution. Urea ((NH2)2CO) and NPK (nitrogen-phosphorus-potassium)
are nutrients that help yeasts to grow well, improving the yeasts quality and production. The stock was
later added into fermenter with molasses and more distilled water to form 12 litres of working media for
fermentation.

Experimental Design of Process Condition for Bio-Ethanol Production

Experimental design for optimization of process conditions was done using 2 Level Factorial
design. Three factors were considered for optimization; temperature, pH, and agitation speed. Aeration
rate was set to constant at 1 LPM since the reactor cannot control the aeration rate. Numbers of runs of
experiments were generated with different conditions to determine the optimum conditions for bio-
ethanol production. Each factor was varied over 2 levels where it is very useful for estimating main
effects and interactions between factors. 2 Level Factorial design was used in order to reduce numbers of
run during fermentation process in bioreactor. Three parameters were monitored and observed in
fermentation process in order to produce optimum ethanol concentration. Three parameters with one
response had prompted the table with 10 runs including two central points with respective randomized
arrangement of values to eliminate errors. Numbers of runs are feasible to the bioreactor operation since 1
run approximately takes 2 days to complete 1 cycle including sterilization and cleaning process. The
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purpose of designing experiment is to set the bioreactor, 30 litre BioSys fermenter that was operated at the
optimum parameters condition for S.cerevisae. The main goal of this basic research for industrial
application is to reduce the total cost of production by optimizing process conditions (The steps of
production in the bioreactor shown in Figure 3).

Switch on the main power supply

After 15 minutes, start filter

sterilization

Empty tank sterilization

Calibrate pH probe and install it to

the fermenter

Media preparation and sterilization

Set up acid, base, and anti-foam

When T=35°C, inoculate yeasts

Fermentation (1 day)

Cleaning

Figure 3: Bioreactor Operations of Ethanol Production by Fermentation

RESULT AND DISCUSSION

S.cerevisae to Produce Ethanol

The first objective of this study is to optimize the bioreactor operational parameters such as
temperature, pH, and agitation to produce high quantity of bio-ethanol based on molasses. The optimized
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media recommended by previous study [10] was used while aeration rate was kept constant at 1 litre per
minute (LPM). The experiment was carried out in 30 litre bioreactor where it is designed to provide best
possible growth and biosynthesis conditions for industrially important microbial cultures. In fermenter, it
is easier to control operational parameters that increase the ease of obtaining desired product, ethanol in
present study. Molasses were diluted up to 14% (w/v) of initial sugar concentration. The sugar
concentration in molasses must not beyond 20% since it might inhibit the growth of yeast and affect
ethanol production. Three parameters were simultaneously studied, temperature, pH, and agitation speed.
Samples were withdrawn every 6 hours and the fermentation was carried out for 24 hours. After carrying
out the fermentation, the samples were analyzed using GCMS and compared with the standard run of
95% ethanol. From the broadness of peak it was interfered that there was ethanol production till 24 hours.
The study was conducted by 2-Level Factorial Design. It was the best design for this study since it
helps to screen many factors to discover the vital few and describes how they interact. Since the study
was involved in the large-scale production, minimum runs are needed for cost and energy effectiveness. 3
operational factors are thought to influence the ethanol production where at each combination of these
parameters, the experimenters recorded the ethanol production concentration. Referring to Table 1, the
highest production of ethanol was held at the central point; Run 3 where 93.10 g/l of ethanol was
produced. Initial sugar concentration provided by molasses was 140.58 g/l. It shows that 34.77% of sugar
was converted to ethanol. Generally, sugars are converted into cellular energy and thereby produce
ethanol and carbon dioxide as metabolic waste products. Yeasts perform this conversion catalyzed by
zymase that presents in yeasts.

Table 1: Experimental result for Bioreactor Conditions Optimization using Design Expert
Run pH Temp (°C) Agitation [Ethanol]
(rpm) (g/l)
1 4.00 40.00 150.00 77.33
2 5.00 30.00 100.00 83.41
3 4.50 35.00 125.00 93.10
4 4.00 30.00 100.00 77.71
5 4.50 35.00 125.00 92.82
6 5.00 30.00 150.00 90.25
7 5.00 40.00 100.00 82.75
8 4.00 30.00 150.00 83.03
9 5.00 40.00 150.00 84.55
10 4.00 40.00 100.00 76.00

Statistical analysis of variance (ANOVA) is very important to tests the significant differences
between means statistically. It detects interaction effects between variables; generally an effect is
modified by another effect. Effects and interaction between factors were observed and analyzed based on
ANOVA results as shown in Table 2.

Table 2: Results Generated by ANOVA for Bio-ethanol Production from Molasses

Source F-Value Prob > F
Model 290.95 0.0034 Significant
A 1021.22 0.0010 Significant
B 267.80 0.0037 Significant
C 330.18 0.0030 Significant
AB 1.56 0.3384 Insignificant
AC 5.59 0.1417 Insignificant
BC 115.16 0.0086 Significant
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Curvature 2219.85 0.0005 Significant

Residual
Lack of Fit 3.52 0.3119 Insignificant

Ideally, value of “Prob > F” less than 0.0500 indicates model terms are significant. From Table 4.2,
the Model F-value of 290.25 implies the model is significant. There is only 0.34% chance that a “Model
F-Value” this large could occur due to noise. In this case A, B, C, and BC are significant model term. No
model reduction was done in this study since there are only two insignificant models; AB and AC. Lack
of Fit is not significant as the value is 3.52. 31.19% chance that a “Lack of Fit F-Value” this large could
occur due t noise. It is good and the model is fixed.
The prediction of future outcomes on the basis of other related information was done in statistical
model by using coefficient of determination R-squared (R2). For this study, R2 = 0.9989 implies 99.89%
of the sample variation for the ethanol production is attributed to the independent variables, pH,
temperature, and agitation speed. It also indicates that the regression line almost perfectly fits the data
since only 0.11% of the variation is not explained by the model. The goodness of fit of a model cannot be
determined by only one possible R2. Predicted R2 (Pred- R2) was also used in the regression analysis to
indicate how well the model predicts responses for new observations. Pred- R2 = 0.9744 shows the model
has a predictive ability up to 97.44% for new observation. Adjusted R2 (Adj- R2) penalizes the statistics as
extra variables included in the model. Adj- R2 = 0.9954 shows a reasonable agreement with Pred- R2. The
R2 values were very high indicates the model fits very well with a very small probability for overfitting
model. The signal-to-ratio (S/N) of 63.244 indicates an adequate signal where the model can be used to
navigate the design space. Effects and interactions of variables were analyzed from the graph generated
by MINITAB. It used to determine the optimum parameters for maximum ethanol production.
Optimum temperature, pH, and agitation were found to be 35 °C, 4.5, and 125 rpm respectively
result in the maximum production of ethanol, which is almost similar to study done by [11]. It is fit with
the prediction point given by the model. It also can be concluded that that temperature and agitation speed
give greater effect on the production of ethanol compared to pH. Optimized temperature exerts a profound
effect on growth, metabolism, and survival of the yeast. It maintains high cell viability resulted in high
production of ethanol. It also helps to reduce operational cost in the production of ethanol since minimum
cooling process is needed for fermenter. Minimum heat was evolved during the fermentation process at
optimum temperature resulted in minimum temperature rise in fermenter.
Agitation speed gives an important impact for the successful progress of the fermentation.
Optimized agitation gives uniform mixing of the media components within the fermentor. It helps cells
and a nutrient disperse uniformly as well as provides adequate mass and heat transfer. Besides it
maintains homogenous chemical and physical conditions in the culture by continuous mixing. Optimized
agitation is also minimized the morphological changes in yeasts, variation in their growth and product
formation, and damaging the cell structure due to shear force results in the maximum production of
ethanol.
Optimization pH was done in order to achieve highest production of ethanol from molasses.
Generally, low pH inhibits yeasts multiplication. Optimized pH was found at 4.5 where it shows the
highest production of ethanol. This optimization result similar to some research done by [12].

Validation of Optimized Parameters

Effectiveness of the finding was evaluated through validation process as in Table 3. Three out of
ten possible solutions given by ANOVA were tested in order to prove the model is fit. It is also the key to
instill the confidence in a building simulation tool. Interactions and effects of factors are explained
algorithmically through equation 1 generated by ANOVA as below:

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Ethanol (g/l) = 34.0763 + 2.3975*pH + 0.5483*Temperature (°C) + 0.3030*Agitation (rpm) +

0.0525*pH* Temperature (°C) + 0.0199*pH*Agitation (rpm) – 0.0090* Temperature (°C)*Agitation
(rpm). (1)

Table 3: Validation Result

Run pH Temperature Agitation Actual EtOH Predicted EtOH
('C) (rpm) (g/l) (g/l)
1 4.4 39.0 132 80.04 80.08
2 4.7 34.5 141 84.71 84.75
3 4.3 40.0 124 78.78 78.73

From the Table 3, there are two values of ethanol concentration, actual and predicted. Actual value
was obtained from the experiment while predicted value was generated by the model equation. Both
values were close to each other indicating the findings are effective. The regression value between actual
and predicted is 99.95% implies the model and findings are reliable.

CONCLUSION

Sugar cane molasses was fully utilized into useful materials for ethanol production. It is abundance,
cheap, and eco- friendly where it produced ethanol; renewable energy to substitute and/or complement
fossil fuels sources. Community dependency on petroleum will be reduce with the existence of bio-
ethanol hence instability prices of petrol in the oil market can be overcome. This study also helps in
reducing emission of CO2 where bio-ethanol is generally CO2 neutral. It gives cleaner burning
characteristic and reduces the formation of CO. Since bio-ethanol is a renewable, agricultural-based raw
materials it can replaces globally depleting hydrocarbon sources like fossil fuels.
The optimum process conditions enhance bio-ethanol production from sugar cane molasses as the
major media. The validation of data was described in the model equation that relates % of ethanol
production with temperature, pH, and agitation speed. Overall, production of bio-ethanol by sugar cane
molasses as substrate offers beneficial alternative for environment in terms of fully utilizes by-products in
to beneficial materials. It also provides minimum cost of total production where substrate used is cheap
and abundance and optimum process conditions minimize the usage of power and energy in the
production process.
Process parameters were successfully optimized at pH 4.5, 125 rpm, and 35 °C where 9.31% of
ethanol was produced. High yield of bio-ethanol, 9.31% was achieved in this study since it was conducted
in BioSys 30 l bioreactor where all process conditions were monitored and controlled. 34.77% of sugar
was converted to ethanol by yeasts. The results would not be the same if the experiment is conducted in
other type and size of bioreactors since the velocity of each reactor is not linear. Height-to-diameter ratio
of bioreactor and the velocity of reactor must be linear in order to get the same production; where it is
found to be quite impossible. The production of ethanol is expected to be higher when mutant-industrial
yeasts are used for fermentation. Mutant yeasts have higher efficiency on sugar utilization and produce
ethanol up to 20% as they have higher tendency to live in high concentration of ethanol compared to the
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wild type yeasts. But, it is not recommended to use mutant yeasts as the mutation is unpredictable and
uncontrollable. They have higher possibility to mutate at unfavourable sites; it is very risky and harmful
to environment and human being.
Model was validated and it was proven that the model is fit and reliable as the regression value
between predicted value and actual value of ethanol concentration is 99.95%. Besides, the study agrees
that the production of bio-ethanol by S.cerevisae from molasses is feasible and applicable for industrial
practice.

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