 Influence of the D-glucose concentration on the fermentation process
.
Influence of the D-glucose concentration on the
fermentation process
Bulthuis, L.C.M., Eggenkamp, M.W., Verdam, M.C.
.
Team GGH26
Gemeentelijk Gymnasium Hilversum, The Netherlands
March 2010
Abstract
One of mankind’s most used fuel and oil will be exhausted in the future. Because of this, interest has grown in
an old chemical process: the production of bio-ethanol out of sugars by fermentation. Yeasts are eukaryotic
micro-organisms, with about 1500 species known and described. They can ferment glucose into ethanol and
carbon dioxide. This will happen when there is no oxygen in the environment. This raises the question of
what the optimal concentration D-glucose will be in the conversion of glucose to ethanol by yeast cells. The
fermentation progress was followed at different solutions, following amounts of D-glucose were dissolved:
1 g, 2 g, 3 g, 4 g, and 5 g. This results in a process that will be more efficient with a higher amount of Dglucose in the solution.
Introduction
Several laboratories and industries use synthetic
ethanol. They use ethene to produce ethanol. The
main source of ethene is crude oil. This production
of synthetic ethanol may become a problem when
crude oil will run short. Synthetic ethanol is now
chiefly produced by the reaction between ethene
(g) and water (g): CH2=CH2 (g) + H2O (g) 
CH3CH2OH (g)
There must be another way to produce ethanol
without the use of crude oil. One of the oldest
chemical processes to produce synthetic ethanol is
the production of ethanol from sugars by
fermentation. The main source of sugars for
fermentation is starch. Starch is made with the
influence of sun. The energy of the sun is used to
synthesize sugars. In this process, atmospheric
carbon dioxide and water are used to produce
glucose: sunlight
6CO2 (g) + 6H2O (g) ------ C6H12O6 (s) + 6O2
(g)
Saccharomyces cerevisiae can invert the C6H12O6.
Yeast cells of Saccharomyces cerevisiae are used in
research to increase the yield of the production of
bio-ethanol from sugars. Yeast does not require
sunlight to grow, but they use the sugars as source
of energy. This yeast uses two major pathways for
growth on glucose:
(1) The fermentation of glucose:
C6H12O6 (s) 2CH3CH2OH (l) + 2CO2 (g)
(2) The oxidation of glucose:
C6H12O6 (s) + 6O2 (g)  6CO2 (g) + 6H2O (l)
These pathways show us that S. cerevisiae cells can
grow in an oxygen free environment and an oxygen
rich setting. The first pathway is the most
interesting one, because we were looking for a way
to replace the crude oil as a source of ethene. But
which is the cheapest way to produce ethene using
S. cerevisiae? This raises the following question: In
which condition, considering the concentration of
glucose, will yeast cells produce the most ethanol?
There can be another way. The enzyme invertase is
found in yeast, which is used to convert
disaccharides and polysaccharides to glucose and
other stereoisomers of C6H12O6. Yeast cells of
Beside ethanol carbon dioxide is produced. The
more ethanol is produced, the more carbon dioxide
is released. We expect the amount of released
carbon dioxide will be the highest in the third tube.
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 Influence of the D-glucose concentration on the fermentation process
This is the tube with an average concentration of
glucose. The activity of enzymes is determined by
several factors, including the amount of ethanol.
Ethanol is one of the substrates of the fermentation
of glucose. The more glucose will be inverted, the
more ethanol will be produced. In the end there is
so much ethanol that it will have a negative
feedback on the enzymes. This means, the yeast
cells activity will be reduced or stop. With a little
amount of ethanol as substrate, there will not be
any negative feedback, which we expect to happen
in the first 3 tubes. But we expect the amount of
ethanol to have influence on the enzyme activity in
the fourth and fifth tube.
Experimental procedure and
approach
Within a day we measured the flasks again. Table 1
presents, in duplicate, the mass (in grams) of the
released CO2 gas at 1, 2, 3, 4, and 5 g D-glucose
solution.
Moreover, it presents the averaged masses of the
released CO2 (in grams) and its deviations at the
various % of D-glucose.
D-glucose (g)
Mass CO2 (g)
1
0.15
0.13
0.28
0.27
0.00*
0.41
0.60
0.51
0.60
0.61
2
3
4
5
Averaged Mass
CO2 (g)
0.14 ± 0.01
0.275 ± 0.005
0.41
0.555 ± 0.045
0.605 ± 0.005
Table.1. Release of CO2 (in grams) and averaged
release of CO2 at 1, 2, 3, 4, 5 g D-glucose.
* The mass CO2 is 0.00. This result shows that the
process did not occur.
Release CO2
Averaged Mass CO2 (g)
5 D-glucose solutions were prepared in duplo. In
30mL demi-water the following amounts of Dglucose were dissolved: 1 g, 2 g, 3 g, 4 g, and 5 g.
For the yeast solution a tablet of S. cerevisiae was
dissolved in demi-water. After that all test tubes
were filled with the D-glucose solutions and 1 mL S.
cerevisiae dissolved in demi-water. All the test tubes
were closed with a hydrogen stop (figure 1). Now
the mass of the test tubes was measured. The
labeled test tubes were placed in a water bath of 37
°C. After one day the test tubes were measured
again. Before the tubes were measured it was made
sure that the reaction in the tube was completed.
Reaction is completed when all the yeast cells are at
the bottom of the tube and no bubbles are seen.
Results
0,7
0,6
0,5
0,4
0,3
0,2
0,1
0
0
2
4
6
D-glucose (g)
Fig.2. The averaged measured release of CO2 (g) at
the D-glucose solutions of 1, 2, 3, 4, and 5 g.
Fig. 1. hydrogen stop
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 Influence of the D-glucose concentration on the fermentation process
Data analysis
As shown in table 1, the average maximum amount
of released CO2 (g) in the fermentation process is
0.605 g. This is equal to 0.605g/44.01u = 0.014 mol
CO2. The first equation shows that in theory 2.00
moles of CO2 gas can be produced. The
percentages of the produced moles CO2 in our
experiments are:
1. 29%
2. 28%
3. 28%
4. 28%
5. 25%
Hereby the calculation of the percentages of the
produced moles CO2 in our experiment.
First, we divided the Molair Mass of CO2 by the
grams of CO2:
1. 0,14/44,0 = 0,0032 mol
2. 0,275/44,0= 0,00625 mol
3. 0,41/44,0= 0,0093 mol
4. 0,555/44,0=0,0126 mol
5. 0,605/44,0= 0,01375 mol
After this calculation, we divided the grams of Dglucose by the Molair Mass of Glucose and we
multiplied the result by 2, because the proportion
between D-glucose and CO2 is 1:2.
1. 1/180,2 x 2 = 0,01109 mol
2. 2/180,2 x 2 = 0,02219 mol
3. 3/180,2 x 2 = 0,03329 mol
4. 4/180,2 x 2 = 0,04439 mol
5. 5/180,2 x 2 = 0,05549 mol
After this, we divided the moles CO2 by the moles
D-glucose and multiplied the result by 100%:
1. 0,0032/0,01109 x 100%= 29%
2. 0,00625/0,02219 x 100%=28%
3. 0,0093/0,003329 x 100%=28%
4. 0,0126/0,04439 x 100%=28%
5. 0,01375/0,5549 x 100%=25%
As shown in figure 2, the averaged maximum
amount of released CO2 (g) in the fermentation
process is 0.605 g. The form of the figure shows
that the process will be more successful if the
concentration of D-glucose is more than 1 or 2 g
D-glucose. The line is less straight when the
concentration of D-glucose becomes too high.
Conclusion, discussion and
evaluation
As shown in table 1, the CO2 gas production was
highest at a 5g D-glucose solution. The 0.605 g CO2
or 0.014 mol of CO2 gas produced less than the
0.56 moles that can be produced. The proportion
between D-glucose and CO2 is 1:2. 5g D-glucose or
0.028 mol can produce 0.056 mol of CO2.
Looking critically at our results will show that one
of our results is not in line with the others. The
weight did not increase, so the process has not
occurred. Therefore we have decided to exclude
this value in our data.
Something must have been present in this sample
that prevented the reaction to occur.
When we took a look at the test tubes, it seems
that all the reactions were completed.
There were no more bubbles in the test tubes and
the yeast descended to the bottom of the test
tubes. Apparently something happened with the
first test tube which had 3 g D-glucose in our
experiment. We think this is strange, because both
test tubes were prepared the same way. We have
the opinion nothing went wrong during the
preparation of the test tubes.
As we have said in our hypothesis, the forming of
CO2 will increase when the concentration Dglucose in the solution is higher. We also predicted
a decrease in the graphic (figure 2) caused by an
excessive concentration of ethanol. However, this is
not shown in our graphic. This will not say we are
wrong.
When there is a bigger difference between the Dglucose concentrations, there is a possibility that
the decrease is shown in our graphic, because there
is more ethanol produced.
However, you can see that the line in the graphic
inclined less when the concentration of D-glucose
became higher. As mentioned, we have said in our
hypothesis that we would see a parabola. If we
would repeat the experiment, we will increase the
amount of D-glucose in the beginning. There would
be more ethanol produced, which would have a
negative feedback on the enzyme activity.
Bibliography
-
www.bioplek.org The influence of ethanol
on the activity of enzymes.
http://en.wikipedia.org/wiki/Ethanol_fermen
tation
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