Alternative production of ethanol by fermentation
Introduction
Nowadays ethanol for industrial and laboratory use is chiefly produced by the reaction between
ethane (g) and water (g):
C2H6(g) + H2O(g)  CH3CH2OH
This method of ethanol production will likely cause a worldwide problem when the crude oil, the
main source of ethane, will run short. Sources are being depleted in great rate. The American
Petroleum Institute estimated in 1999 the world's oil supply would be depleted between 2062 and
2094. This has stimulated interest in the investigation of another reliable ethanol production. One
of the most prominent occurring investigation concerning this subject is: the production of
ethanol by fermentation.
Mono-sugars like D-glucose are fermentable into ethanol. D-glucose has several stereo isomers.
Disaccharides and polysaccharides must be converted to fermentable sugars (mono-sugars) by
hydrolysis. Sucrose, a disaccharide, is hydrolyzed by an enzyme found in yeast.
The yeast species Saccharomyces cerevisiae has been used in baking and fermenting alcoholic
beverages for thousands of years. It is extremely important as a model organism in modern cell
biology research, and is one of the most thoroughly researched eukaryotic microorganisms.
Yeast cells of S. cerevisiae are commonly used during the process of fermentation.
Yeasts are chemoorganotrophs; organisms which use organic compounds as their energy source.
These organic chemicals include glucose. Therefore Yeasts do not require sunlight to grow.
Yeasts grow best in a neutral or slightly acidic pH environment. Yeast grow best in an
environment that is controlled at 20-40°
Saccharomyces cerevisiae cells use three major pathways for growth on glucose.
1. fermentation of glucose
C6H12O6(s)  2CH3CH2OH(l)
2. oxidation of glucose
C6H12O6(s) + 6O2(g)  6CO2(g) + 6H2O(l)
3. oxidation of glucose
CH3CH2OH(l) + 2O2(g)  2CO2(g) + 3H2O(l)
The fermentation of glucose is interesting for our research, seeing it involves producing ethanol.
Starch or amylum (C6H10O5)n is a HYPERLINK "http://en.wikipedia.org/wiki/Carbohydrate" \o
"Carbohydrate" carbohydrate consisting of a large number of HYPERLINK
"http://en.wikipedia.org/wiki/Glucose" \o "Glucose" glucose units joined together by
HYPERLINK "http://en.wikipedia.org/wiki/Glycosidic_bond" \o "Glycosidic bond" glycosidic
bonds (figure 1). These alpha bonds are easily hydrolyzed. This polysaccharide is produced by
all green HYPERLINK "http://en.wikipedia.org/wiki/Plant" \o "Plant" plants as an energy store.
Starch is processed to produce many of the sugars in processed foods. It is contained in foods as
HYPERLINK "http://en.wikipedia.org/wiki/Potato" \o "Potato" potatoes, HYPERLINK
"http://en.wikipedia.org/wiki/Wheat" \o "Wheat" wheat, HYPERLINK
"http://en.wikipedia.org/wiki/Maize" \o "Maize" maize and HYPERLINK
"http://en.wikipedia.org/wiki/Rice" \o "Rice" rice. Pure starch is a white, tasteless and odorless
powder that is insoluble in cold water or alcohol.
Glucose is soluble in water, glucose in the form of starch, on the other hand, is not soluble in
cold water. Starch becomes soluble in water when heated. The granules swell and burst, the
semi-crystalline structure is lost and the smaller amylase molecules start leaching out of the
granule, forming a network that holds water and increasing the mixture's HYPERLINK
"http://en.wikipedia.org/wiki/Viscosity" \o "Viscosity" viscosity. When dissolved in warm water,
it can be used for thickening, stiffening or gluing.
HYPERLINK "http://en.wikipedia.org/wiki/File:Amylose2.svg" INCLUDEPICTURE
"http://upload.wikimedia.org/wikipedia/commons/thumb/2/21/Amylose2.svg/270pxAmylose2.svg.png" \* MERGEFORMATINET
Figure 1: amylum
Starch makes up a large proportion of our diet, and it needs to be made soluble before it can be
absorbed into our bodies. In order
to carry out this chemical breakdown, various glands produce digestive juices containing
amylases to be mixed with the
food. Saliva contains one amylase, and pancreatic juice contains another.
Quantity of amylum(gr)
Quantity H2O (ml)
Test tube 1.
0,25
100
Test tube 2.
0,50
100
Test tube 3.
0,10
100
+ 1ml spittle + 0,20gr yeast Saliva is the watery substance produced in the mouths of HYPERLINK
"http://en.wikipedia.org/wiki/Human" \o "Human" humans and most other HYPERLINK
"http://en.wikipedia.org/wiki/Animal" \o "Animal" animals. Human saliva is composed of 98%
HYPERLINK "http://en.wikipedia.org/wiki/Water" \o "Water" water, while the other 2%
consists of other compounds and various HYPERLINK "http://en.wikipedia.org/wiki/Enzyme"
\o "Enzyme" enzymes. As part of the initial process of food digestion, the enzymes in the saliva
break down some of the starch and fat in the food at the molecular level. The enzyme that we are
interested in is amylase which breaks down starch into sugar. Amylase breaks down long-chain
carbohydrates along the starch chain. This will yield glucose and other sugars. Its optimum pH is
6.7-7.0.
If we were to use the enzyme amylaze to create glucose from amylum would the glucose suffice
in the process of creating ethanol by yeast cells. Seeing amylum consist of a large chain of
glucose molecules this would perhaps produce a large amount of glucose. Perhaps it would even
be cheaper or more sufficient than using the obvious source: (table)sugar.
This raises the inquiry question
Will saccharomyces cerevisiae produce ethanol utilising the glucose units broken down from
amylum?
Our hypothesis is that as long as the experiment is performed in an environment with a
temperature friendly towards the yeast cells ethanol will be produced. Seeing the amylum will
not entirely break down in glucose, not the entire solution will be used by the yeast cells.
Experimental procedure and approach
We prepared 3 solutions of amylum(aq). the water temperature was +- 20-30 degrees, seeing that
starch can only be dissolved in warm water. Each solution contains 100 ml water and a various
amount of starch; 0.25, 0.5, 1 gram.
Table 1: experimental procedure
Next we add the enzyme amylase in the form of spit. in each test tube we add
approximately 1 ml of spittle to each test
tube. Next we add to each test tube 0,2grams of yeast. Then a bottle filled with water, of which
the mass was measured beforehand, was fit over the test tube with a hosepipe. The bottle filled
with water was placed in a container with water in such a position that no air could escape the
bottle or get in. as a result the created gas would travel through the pipe into the bottle. The
quantity of gas can be measured by the difference in weight of the bottle after the experiment.
The quantity of created ethanol CH3CH2OH (mol) can calculated out of the quantity of carbon
dioxide CO2 (mol). Throughout the experiment the temperature must remain 25 degrees Celsius
(this because of the law of gas).
Figure 2: experimental setup.
Experimental Results
Full bottle of water: 565grams
Table 2
0,25g/100mL amylum
time (hours)
48
Weight bottle
562,18
96
558,59
Table 3
0,5g/100mL amylum
time (hours)
48
Weight bottle
554,29
96
552,38
Table 4
1g/100mL amylum
time (hours)
48
Weight bottle
550,6
96
547,91
Reaction between glucose and yeast cells:
C6H12O6 + yeast cells  2 C2H5OH + 2 CO2
1
+.
2
+2
Table 5
time(hours)
0
48
96
0,25g/100mL amylum
Mass bottle (g)
565
562,18
558,59
difference (565 – x)
0
2,82
6,41
(x, weight of bottle versus time(units))
Table 6
time(hours)
0
48
96
0,50g/100mL amylum
Table 7
time(hours)
0
48
96
1,00g/100mL amylum
Mass bottle (g)
565
554,29
552,38
Mass bottle (g)
565
550,6
547,91
Table 8
0,25g/100mL amylum
time(hours))
0
difference (565 – x)
0
10,71
12,62
Difference (565 – x)
0
14,40
17,09
Volume H2O (l)
0
48
96
2,83*10-6
6,42*10-6
(measurements table 4, 5 & 6 *( /density water))
Table 9
time(hours)
0
48
96
0,50g/100mL amylum
Table 10
time(hours)
0
48
96
1,00g/100mL amylum
Table 11
time(hours)
0
48
96
0,25g/100mL amylum
Volume H2O (l)
0
1,07*10-5
1,26*10-5
Volume H2O (l)
0
1,44*10-5
1,71*10-5
CO2 (mol)
0
0,000115
0,000262
(measurements, table 7, 8 & 9 (molair volume;0,0245))
Table 12
time(hours)
0
48
96
0,50g/100mL amylum
Table 13
time(hours)
0
48
96
1,00g/100mL amylum
Table 14
time(hours)
0
48
96
0,25g/100mL
CO2 (mol)
0
0,000438
0,000516
CO2 (mol)
0
0,000589
0,000699
Methanol (g)
0
0,005313
0,012077
(measurements table 10, 11 & 12 (molmass; 46,068))
Table 15
time(hours)
0
48
96
0,50g/100mL amylum
Table 16
time(hours)
0
48
96
1,00g/100ml amylum
Methanol (g)
0
0,020179
0,023777
Methanol (g)
0
0,027131
0,032199
figure 3: amount ethanol versus time
Data analysis
As presented in figure 2 the amount of starch influences the produced amount of ethanol.
The curve of line (0,225gram/100ml) is different from the other two lines, this can be explained
be the fact that not al the glucose units have reacted yet. Be analyzing figure 3 it becomes clear
that the increasing amount of starch increases the speed of the ethanol producement.
Conclusion and discussion
The observation that all the bottles lose weight indicates that in all bottles the yeast cells were
growing and produced CO2 gas and therefore also ethanol. As shown in fig. 4. the release of
ethanol was greatest with the highest amount of amylum.
However we have not registered the correct amount of amylase in the saliva, nor the approximate
amount of saliva. These control variables can be measured more precisely by using an amylase
solution instead of saliva. Now we do not know the correct measure of influence of amylase.
This raises a further question for inquiry: what is the most effective amount of amylase.
The lines in figure 4. do not go the way we expected. Looking critically at our experimental
procedure and approach we see that in all sets of experiments we considered the same
independent and dependent variables and we kept the same variables constant. We can conclude
that this difference is explained by the measurement uncertainty.
Line 3 ( 1gr/100ml) is supposed to decline increasing, this is also explained by the measurement
uncertainty.
bibliography
HYPERLINK "http://nl.wikipedia.org/wiki/Gist" http://nl.wikipedia.org/wiki/Gist
Joost Groen & Pieternel Kims
klas 5
12 April 2010
Time (hours)
Shaping ethyl alcohol/hour