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Bioethanol production by Enzymatic Hydrolysis of Cellulose using

immobilized Saccharomyces cerevisiae in fedbatch Fermentation:
A Kinetic study.
Ahmad Mohammed Gumel,
Biotechnology Unit, Institute of Biological Sciences, University of Malaya, 50603, Kuala Lumpur, Malaysia

Abstract:
The purpose of this study was to highlight the enzymatic kinetics in the hydrolysis of cellulose for
Bioethanol production. Immobilized Saccharomyces cerevisiae was used as fermentative organism,
while Carboxymethylcellulose (CMC) was used as fermentation substrate. Different substrate
concentrations were used; parameters such as enzymatic velocity, substrate concentration, glucose
concentration, bioethanol yield and productivity were studied under 15 minutes fermentation time.
Under optimized condition of 10g/l CMC with glucose hydrolysis concentration of 0.166g/l
bioethanol production was found to be 42% (w/v) at 15 minutes of fermentation time. Langmuir and
Michaelis-Menten models were used to plot and validate the experimental data respectively.

1. Introduction:
Biofuels such as bioethanol, biohydrogen and biodiesel are anticipated to be one of the feature
alternatives to fossil fuels. The current world consumption of 474 exajoules (5×1020 J) with 80 to 90
percent derived from the combustion of fossil fuels1, which are associated with production
instability, insecurity, sky rocketed prices as well as green house gasses emissions; are among the
reasons that lead policy makers to seek for energy alternatives that are sustainable and environmental
friendly.
Today bioethanol is among the most widely employed biofuel in the world with current production
of about 19 billion gallons world wide, in United State alone it provides about $12.3 billion capital
market, and creates 238,541 jobs as at 2007 2, bioethanol production is expected to pass over 20
billion gallons by 2012.
Although, food-crops are known to be the main feedstock in bioethanol production, the use of post
harvest agro-waste and non-food crops cellulosic materials are highly encouraged, this is because of
the food security issue, though there are surplus amount of food in some countries, millions of
people in other countries most especially developing countries face food shortage scenario.
Therefore extensive used of food crops such as corn, wheat, soybeans, palm oil as a feedstock in
bioethanol production, may result in a serious food security problems.
Cellulosic resources, such as agricultural residues, paper wastes and wood chips, are the most
abundant organic substance in nature and considered to be promising and economically feasible
feedstock for biofuel production 3. Enzymatic hydrolysis by cellulolytic enzymes that naturally
degrade these cellulosic materials to monomeric sugars that are fermentable by Microbes are often
required4. Although cellulose may be hydrolised by non- enzymatic methods, i.e. by acid hydrolysis,
the advantages and utility cost of enzymatic hydrolysis are better and lower compared to the acid
hydrolysis. A number of bioethanol production processes that used microorganisms have been
studied to produce ethanol from cellulosic materials. Among them, the stirred tank fedbatch
fermentation process was cited by many researchers5-10.
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Although, several microorganisms were employed in bioethanol production process, but direct
bioconversion of cellulosic materials for example by Clostridium thermocellum is not attractive
because of low ethanol productivity and high energy requirements compared to ethanol production
by Saccharomyces cerevisiae using molasses 11. S. cerevisiae is a yeast, which is believed to be
among the most effective lignocellulosic biomass degrading microorganisms for hexose sugars such
as glucose, mannose and galactose, it has a capability of rapid rates of glycolysis and ethanol
production under optimal conditions, producing over 50 mmol of ethanol per h per g of cellulose 12.
However, this high rate is maintained for only a brief period during batch fermentation and declines
progressively as ethanol accumulates in the surrounding broth 12.

To date numerous research studies regarding the fermentative activities of S. cerevisiae in stirred
tank reactors utilizing cellulosic biomass have been reported, alas! There are limited citations on
kinetic studies of the process; which we believed will help in understanding the overall process for
maximum optimization.

2. Materials and Methods

2.1 Fermentation media and Culture
The substrate used for this study is carboxymethylcellulose (CMC) from laboratory stock to serve as
the cellulose substrate, S. cerevisiae that was used was also obtained from laboratory stock. While
chemicals and reagents such as sodium alginate, calcium chloride, yeast extract, ammonium sulfate,
potassium phosphate, hydrated magnesium sulfate, hydrated calcium chloride, DNS reagents,
Rochelle salt solution reagents were all obtained from Merck and are of pure quality.
2.2 Microbial culture and Immobilization
The strain was subcultured on basic media containing yeast extract 3gl -1, (NH4)2SO4 2.7 gl-1, KH2PO4
2.3 gl-1 , MgSO4.7H2O 0.7 gl-1, CaCl2.2H2O 0.1 gl-1 , which was incubated at 30oC for 24 hours.

5ml hypodermic syringe was used to pour 100ml of (1% v/v) sodium alginate containing the
cultured yeast into a beaker containing 200ml of 0.75% w/v calcium chloride, in a drop wise fashion.
The solution in the beaker was then discarded and replaced with 0.11% w/v calcium chloride and
was incubated at 4oC for an hour. And the beads were filtered for fermentation process.

2.3 Standard Glucose Assay

From the standard glucose stock solution, Different samples concentrations (0.1 gl -1,0.2 gl-1,0.3 gl-
1
,0.4 gl-1,0.5 gl-1), were made; following standard DNS glucose assay protocols by Miller 13, the
spectrophotometric absorbance (OD575nm)of each sample was recorded and plotted against each
concentration, which formed the glucose standard curve.

2.4 Enzymatic Hydrolysis

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19ml of acetate buffer (100mM, 4.6pH) was measured into reaction vessel; the following volumes of
the samples concentration were used and top up with distilled water, which formed final volume of
247.5ml as presented in the table below:
Concentration (gl-1) CMC stock Distilled water Total mixture
volume (ml) volume(ml) volume (ml)
6 75 152.5 247.5
8 100 147.5 247.5
10 125 122.5 247.5

the mixture was homogenized by stirring at 200rpm for 2 minutes. 3ml of each sample was
withdrawn and glucose concentration was assayed as the initial glucose concentration.
2.5ml of cellulase solution was added to each sample while stirring at 200rpm, 3ml sampling was
done every 3 minutes for 15minutes and analyzed for glucose assay13 and pH. The
spectrophotometric absorbance of each sample was recorded and the concentration was determined
using the glucose standard curve.
The final glucose sampling was taken at 24hours of fermentation, was diluted to specified
concentrations and analyzed for glucose assay and pH. Base on the data collected the below tables
were formulated:
Table1: Glucose standard solution Absorbance base on concentration
Concentratio Absorbance
n (gl-1) OD (575nm)
0.1 0.25
0.2 0.4915
0.3 0.736
0.4 0.893
0.5 1.1855

Fig1: Glucose Standard curve

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Result and Discussion:

The production of bioethanol under different CMC concentrations is shown in figure 6. the
bioethanol concentration increased with increased fermentation time, however it was found in all
concentration experimental run to decrease at the end of fermentation time. Each experimental
sampling was run in duplicate to carried out the enzymatic hydrolysis of cellulose for ethanol
production using Saccharomyces cerevisiae in fedbatch fermentation using stirred tank reactor.

At CMC concentration of 6g/l the highest glucose concentration produced by hydrolysis is found to
be 0.085g/l at 9 minutes of experimental time, while in 8g/l and 10g/l CMC concentration, the
highest glucose produced is 0.085g/l and 0.167g/l at 15 minutes of fermentation time respectively.
The concentration of total glucose produced by enzymatic hydrolysis of the CMC as shown in
figure 2-4; was found to fluctuate throughout the fermentation period, however this also indicates a
significant reduction in glucose concentration within the fermentation media towards the end of
fermentation time, a more clear picture of the glucose concentration by enzymatic hydrolysis can be
seen when compared on a single chart as depicted in figure 5.

Figure 2: Glucose concentration by hydrolysis of 6g/l carboxymethylcellulose (CMC)

over different experimental time in the course of the fedbatch fermentation
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Cellulosic

0.14

0.12
Figure 3: Glucose concentration by hydrolysis of 8g/l carboxymethylcellulose (CMC)
over different experimental time in the course of the fedbatch fermentation

0.1
glucose conc (g/l

0.08

Figure 4: Glucose concentration by hydrolysis of 10g/l carboxymethylcellulose (CMC)

over different experimental time in the course of the fedbatch fermentation

0.06
The kinetic parameters such as the initial enzymatic velocity (v) given by δP / δt was found to
varied within the experimental runs of different concentrations; it was found to be 0.0192g[P]/min in
6g/l CMC concentration (figure2),while 0.0205g[P]/min, 0.0231g[P]/min in 8g/l and 10g/l CMC
concentrations respectively (figure3 and 4). We noticed that increased in enzymatic concentration
leads to an increased in velocity and conversion rate, however, this increase tends to stabilize when
the maximum velocity is approach, so to optimized the production process the maximum velocity
range in relation to substrate concentration has to be maintained.
0.04
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[S ] Km [S ]
Therefore, using Langmuir Transformation, : = + ,
[V ] V max V max
[S ] 1
By plotting against [S] in Langmuir plot the slope denoted by ,
v V max
From the experimental data plotted on Langmuir model; X-intercept was used to determine the
Km
negative value of Km, therefore Km = 4.8, hence this was used in Y-intercept equation as
V max
gave the value of Vmax to be 0.034g[P]/min. while individually base on the data collected and
computed the enzymatic maximum velocity (Vmax) within CMC concentrations of 6g/l, 8g/l and 10
g/l was found to be ranging from 0.03-0.034g[P]/min. these values were further confirmed
V max[ S ]
theoretically using Michaelis-Menten equation of V= and were found to be almost
[ S ] + Km
similar with difference of 0.17 in Km value.

When the reaction stoicheometric equation is considered, following the fedbatch fermentation of 15
minutes period using CMC concentrations of 6g/l, 8g/l and 10g/l shown in figure 6, bioethanol
concentrations were found to be 7.56E-3 g[P]/min, 6.9E-3g[P]/min and 5.6E-3g[P]/min respectively.
The productivity value (P = amount of product produced per unit time) in g[P] was found to be
5.04E-4, 4.6E-4 and 3.73E-4 respectively. It is worth noting that the highest ethanol productivity
shown in figure 7, is at CMC concentration of 10g/l with about 42% (w/v) productivity.
When the initial glucose concentration of 38.90g/l is considered, the final glucose concentration after
24 hours fermentation was found to be 6.30 g/l, the total glucose consumed is 32.6g/l. hence total
bioethanol concentration was found to be 16.66g/l therefore the total bioethanol productivity of the
( E max − Eo )
operational mode P = within 24 hours fermentation period was 0.69gL-1h-1.
t
Ethanol production concentration

8.00E-03
ethanol concentration

6.00E-03
g[P]/min

4.00E-03

2.00E-03

0.00E+00
0 2 4 6 8 10 12
CMC concentration in g/l

Figure 6: Ethanol production concentration in g[P]/min using different

CMC concentration as fermentation substrate
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Bioethanol productivity from fedbatch

fermentation using the following CMC
concentration 6g/l,8g/l,10g/l at different
experimental runs
6
10 25%
42%

8
33%
Figure 7: Ethanol productivity in g[P] based on fermentation substrate (CMC concentration)
used within 15 minutes fermentation time.

Conclusions:
Bioethanol production by Fedbatch Fermentation of Hydrolyzed Carboxymethylcellulose (CMC)
using Saccharomyces cerevisiae was achieved using different CMC concentrations, with highest
production of 42% (w/v) in experimental running of CMC 10g/l concentration in 15 minutes, this
process is an attractive process for producing sustainable cost effective and environmental friendly
energy source.
From the result discussed so far, leads to the conclusion that fermentation process can be optimized
when kinetic parameters such as substrate concentration [S], enzymatic velocity (v) are carefully
designed and monitored, for instance the enzymatic velocity has to be carefully designed and
controlled to be within the range of maximum value (Vmax), in order to achieve maximum yield
and maintain production cost as well as enzymatic speed, because once the maximum enzymatic
velocity is reach, increased in substrate [S] concentration will do little or no effect at long last,
resulting in none efficient production process.