Journal of Energy, Environment & Carbon Credits

Volume 2, Issue 3, December 2012, Pages 1-6

__________________________________________________________________________________________

Effect of Sodium Alginate Concentration on Size of Yeast Immobilized Particle

and on Production of Bio-ethanol in a Packed-Bed Fermenter

M. Selvaraj, B. S. V. S. R. Krishna*
Associate Professor, Department of Chemical Engineering, Manipal Institute of Technology, Manipal
University, Manipal-576104, India

ABSTRACT

Biofuels are important because they replace the petroleum fuels or fossil fuels and especially ethanol
and butanol contribute greatly to the mitigation of greenhouse gases emission, provide clean and
sustainable energy source and reduce the consumption of crude oil. Current ethanol production
processes using agricultural crops such as sugarcane and corn are well known, i.e., biochemical
pathway and thermo-chemical pathway. However, in the biochemical pathway, the reactors used for
fermentation have to be improved from a classical well-mixed reactor such as batch fermenter and fed-
batch fermentor to the continuous reactors due to the following reasons, i.e., product inhibition and
wash out, etc. An attempt has been made to improve the reactor design using continuous packed-bed
reactor with baker’s yeast as microorganism and this was immobilized in calcium alginate beads
(particles). In the present study, we have conducted experiments to identify the suitable bead size and
suitable concentrations of sodium and calcium alginate solutions for better sugar conversion or more
ethanol yield in batch reactor. The same beads are used for continuous packed-bed study. The results
are compared. The continuous packed-bed reactor gave better conversions with retention time of 5 h.

Keywords: Biofuels, sodium alginate, calcium alginate, immobilization, packed-bed reactor

*Author for Correspondence E-mail: krishna.bandaru@manipal.edu

1. INTRODUCTION including Clostridium bacterium,

Saccharomyces cerevisiae, the well-known
The 1970 energy crisis led many scientists to yeast, and Zymomonas mobilis are suitable
look forward to low-cost, sustainable and candidates to produce ethanol [2].
renewable energy sources such as ethanol and Conventionally, by batch fermentation the
butanol. Ethanol has been described as one of ethanol production can reach a maximum of
the most exotic synthetic oxygen-containing 8% (v/v). Many studies suggested that due to
organic chemical because of its unique the product inhibition, the batch reactor gave
combination of properties as a fuel, solvent poor results [3]. The continuous packed-bed
and as an intermediate raw material for many reactor gives better conversion as compared to
chemicals. Hang et al. [1] extensively batch reactor due to continuous removal of
discussed ethanol production from a liquid via product and byproducts despite product
submerged microbial fermentation. inhibition. The reactor design and
immobilization of yeast on surface of the
In the past decades, microbial ethanol beads/trapping the yeast in the beads are very
production has been focused and considered as important parameters in the design of packed
an alternative fuel for future since fossil fuels beds. The present study aims at increasing the
are depleting. Several microorganisms, concentration of the bioethanol through

ISSN: 2249 – 8621© STM Journals 2012. All Rights Reserved

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 Journal of Energy, Environment & Carbon Credits
Volume 2, Issue 3, December 2012, Pages 1-6
__________________________________________________________________________________________
continuous fermentation of glucose-based
substances and to compare the ethanol
production from batch and packed-bed reactor
using different size of yeast immobilized
beads. The effect of sodium alginate and
calcium chloride solutions on size of the bead
and production of bioethanol was identified
and presented.
2. MATERIALS AND METHODS
Medium preparation: 10% of glucose, 0.2%
yeast extract, 0.1% K2HPO4, 0.07% MgSO4,
7H2O, 0.4% (NH4)2SO4, 0.1 % NaCl was used
for medium preparation. The pH of the solution
was maintained at 4.5 with sulphuric
acid/ammonium hydroxide. The medium was
sterilized for 15 min at 15 psi pressure and
then cooled to room temperature. The baker’s
yeast or S. cerevisiae cells were allowed to
grow till their exponential phase.
2.1. Dry Cell Weight
The inoculum sample was collected at
specified intervals from the reactor into a pre-
weighed centrifuge vial. The broth was
centrifuged and the supernant was discarded.
The vial was dried for 24 h in an oven at
105 °C and its weight was measured. The
difference was given the weight of dry cells.
2.2. Preparation of Immobilized Beads
Sodium alginate solution was prepared by
slowly dispersing the sodium alginate powder
in distilled water in a continuously stirring
beaker. The sodium alginate solution and
ISSN: 2249 – 8621© STM Journals 2012. All Rights Reserved
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prepared S. cerevisiae cell suspension was
mixed suitably to make a uniform solution.
This solution was added drop-wise to the 2%
CaCl2 solution using a pre-calibrated syringe.
Calibration of syringe was done by weighing
the drop weight using a balance. The drops
formed as soft beads. The beads were allowed
to harden in the CaCl2 solution and washed
with saline solution to clean the excess
calcium ions. The immobilized bead size was
measured by using the electronic microscope.
The immobilized beads were stored at 4 °C
until use [4].
3. ANALYTICAL TECHNIQUES
3.1. Glucose Estimation
The glucose estimation was done using the
DNS method as specified in the literature. A
spectrophotometer (at 575 nm) was used to
find the optical absorbance of sample glucose.
The standard plot was prepared with known
concentrations versus absorbance for various
ranges of glucose concentration. The unknown
glucose concentration was estimated using the
standard plot of absorbance and glucose
concentration.
3.2. Ethanol Estimation
Two methods were used for ethanol
estimation, i.e., wet method and gas
chromatography. The gas chromatography was
used for most of the samples and the wet
method was used for some of the samples
analysis. In the wet method, the ethanol
concentration was estimated using dichromate
 Journal of Energy, Environment & Carbon Credits
Volume 2, Issue 3, December 2012, Pages 1-6
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solution and starch as indicator. Chemito make magnetic stirrer as shown in Figure 1. The
GC (GC 8610) with packed-bed column was RBF openings were closed with the help of
used for estimation of ethanol. rubber corks. One for CO2, second one for
sample collection and third one for
4. EXPERIMENTAL temperature was used. Temperature was
maintained constantly with the help of a
Batch studies were conducted in a 500 mL thermostat.
double-neck round-bottom flask (RBF) with

Fig. 1: Schematic Experimental Setup of Batch Reactor with Immobilized Beads.

ISSN: 2249 – 8621© STM Journals 2012. All Rights Reserved

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 Journal of Energy, Environment & Carbon Credits
Volume 2, Issue 3, December 2012, Pages 1-6
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The continuous packed-bed reactor is shown
in Figure 2. The reactor was made with an
outer diameter of 5 cm glass column with
3 mm thickness and a total height of 0.65 m.
The total volume was 1200 mL and working
volume of the reactor was maintained at
765 mL. The flow rate was maintained with
the help of a peristaltic pump. The temperature
was maintained constantly by immersing the
feed solution in water bath and jacket. The pH
of the solution was measured with the help of
H
a p meter. Addition of sulphuric acid to
H
maintain the proper p was done using a
dosing pump near the feed inlet. One can
expect the following reaction can occur in the
fermenter:
C6H12O6  2C2H5OH + 2CO2
The maximum theoretical conversion of
glucose to ethanol is 51% (92 × 100/180 = 51)
on weight basis.
5. RESULTS AND DISCUSSION
5.1. The Growth Curve
The dry cell weight was measured as discussed
in the methods section. The dry cell weight
increased to a maximum of 15.53 g/L on the
fourth day. The growth, mean and death
phases are shown in Figure 3. The batch and
continuous experiments were started on the
third day and completed on the fourth day to
maintain the log mean phase.
ISSN: 2249 – 8621© STM Journals 2012. All Rights Reserved
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5.2. Effect of Bead Size
For the preparation of beads, 1% to 6% of Na
alginate with 2% CaCl2 solution was used. The
soft and gel type of beads were formed with
low concentration of Na alginate solution. The
soft gel changed its shape before usage while
2% to 6% concentration could be suitable for
experimentation. The size of the beads were
also measured and it was found that the size
depends on concentration of Na alginate while
using single aperture syringe. As the
concentration increases, the bead size also
increases and stabilizes at constant size. The
effect of the bead size on Na alginate
concentration is shown in Figure 4. The same
beads were used for batch studies with 500 mL
in RBF. The experiment was conducted for
three days to identify the effect of the bead
size/Na alginate oncentration on glucose
conversion. The results are shown in Figure 4.
The glucose conversion decreased and
increased with Na alginate concentration. The
following phenomenons can be seen with
increase of Na alginate concentration, i.e., (1)
increase in the size of bead and (2) increase in
the hardness of the bead. The increase in size
of the bead will reduce the surface area of the
bead, subsequently reducing the glucose
conversion. The increase in hardness of beads
makes it more difficult for the substrate to
interact with cells.
 Journal of Energy, Environment & Carbon Credits
Volume 2, Issue 3, December 2012, Pages 1-6
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Fig. 3: Variation of Dry Cell Weight with
Time.
Fom Figure 4, one can observe that at 4% Na
alginate concentration, the remaining glucose
percentage was less and particle size also
reseasonable at a diameter of 2.5 mm. This
diameter was neither small nor large for usage
in a packed bed. For maintaining the cell to
substrate concentration ratio, though the
glucose conversion increased with size of bead
and concentration of Na alginate, the present
study prefered 4% Na alginate concentration
and 2.5 mm of bead size for batch
fermentation.
The same phenomena was apllied and
experimentation was continued with 2.5 mm
and 4% Na alginate concentration for
continuous packed-bed study. The hardness of
bead depends on Na alginate concentration
and also on concentration of CaCl2. The
present study focused on the effect of CaCl2
concentration on bead size. The Na alginate
concentration was fixed at 4% and varied the
concentration of CaCl2. The effect was very
small upto 4% of CaCl2 and beyond it, the
ISSN: 2249 – 8621© STM Journals 2012. All Rights Reserved
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Fig. 4: Effect of Na Alginate Concentration on
Bead Size and Glucose Conversion.
effect increased as shown in Figure 5. To
reduce the cost of immobilization, in the
present study, 2% CaCl2 concentration was
chosen for immobilization as discussed by
Kostov et al. [5].
5.3. Packed Bed
The immobilized beads (made with yeast cells,
4% Na lginate and 2% CaCl2 solution) with a
size of dia 2.5 mm were loaded up to three
fourth of the column from the top of the
packed bed reactor. The substrate was
prepared with an initial concentration of
53 g/L and kept in a water bath for
maintaining the temperature and pH was
maintianed with the help of sulphuric acid
and/or ammonium hydroxide. The flow rate of
the substrate was maintained at 5 mL/min with
the help of a peristaltic pump. The substrate
was recycled twice through the packed bed for
maintaining the retention time of 5 h and
glucose and ethanol concentration was
measured. The final glucose concentration was
found to be 25 g/L with a conversion of 52.8%
 Journal of Energy, Environment & Carbon Credits
Volume 2, Issue 3, December 2012, Pages 1-6
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with ethanol concentration of 12.2% (v/v). The
results were compared with Tri Widjaja et al.
[3] and conversion was also better as
compared with the batch reactor.
Fig. 4: Effect of Na Alginate Concentration
and CaCl2 on Glucose Conversion.
6. CONCLUSIONS
Continuous ethanol production in
immobilized cell packed-bed reactor was
succesfully carried our with Na alginate
immobilized beads. Better glucose conversions
were found with 4% Na alginate, 2% CaCl2 at
2.5 mm diameter. The packed-bed reactor was
gave better conversions with respect to the
glucose and ethanol production than batch
reactor at 5 h retention time.
ISSN: 2249 – 8621© STM Journals 2012. All Rights Reserved
ACKNOWLEDGEMENT
The authors are grateful to the Manipal
University for providing the funding for the
project
REFERENCES
1. Hang Y. D, Lee C. Y., Woodams E. E., et
al. Applied Environmental Microbiology.
1981. 42(6). 1128–1129p.
2. Maziar Safaei Asli. African Journal of
Biotechnology. 2009. 9(20). 2906–2912p.
3. Tri Widjaja, Soeprijanto Ali Altway,
Setiyo Gunawan, et al. International
Journal of Academic Research. 2010. 2(6).
30–34 p.
4. Pilkington P. H., Margaritis A., Mensour
an N. A., et al. Journal of The Institute of
Brewing. January-February, 1998. 104.
19–31p.
5. Georgi Kostov, Mihail Angelov, Ivan
Mihaylov, et al. Revue de génie industriel,
2010. 5. 25–35p.
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