Investigation into the Effect of Sugar
Type on Ethanol Production by
Clostridium sporogenes
Julia Bingham
000916-015
May 2012 Exam session
Lakewood High School
Mrs. Sarah Talle
Word Count: 3,610
Bingham 000916-015 2
Effect of Sugar Type on Ethanol Production by C. sporogenes
Table of Contents
Abstract
3
Research Question
4
Aim
4
Hypothesis
4
Background Information
5
Variables
7
Method
8
Processed Data
10
Data Analysis
12
Conclusion
13
Evaluation and Uncertainties
15
Bibliography
18
Appendix
19
Bingham 000916-015 3
Effect of Sugar Type on Ethanol Production by C. sporogenes
Abstract
This paper evaluates an experiment conducted testing ethanol production by the
bacterium Clostridium sporogenes. The research question addressed by this experiment was
‘What is the effect of the type of sugar molecule (glucose or cellulose) on the amount of ethanol
produced by the bacteria Clostridium sporogenes through consumption of the sugar?’
Background research was conducted to reach a hypothesis, which stated that there is an overall
difference in the efficiency of ethanol production based on the sugar molecule used, and that the
bacteria consuming glucose will produce more ethanol than bacteria consuming cellulose or
bacteria in the control environment with no added sugars. In the experiment, cultures of C.
sporogenes were incubated and then added to nutrient broth solutions. Either cellulose, glucose,
or no sugars were added for each trial, and these solutions were left for a period of four days to
allow the bacteria to process the sugars into ethanol. Afterwards, the amount of ethanol produced
in each trial was recorded. The data showed more ethanol produced by bacteria that processed
glucose than cellulose, and no ethanol produced by bacteria that did not have sugar added to the
solution. These results led to the conclusion that ethanol production is dependent on the type of
sugar processed, and that the bacterium C. sporogenes produces ethanol more efficiently when
processing glucose than when processing cellulose.
Bingham 000916-015 4
Effect of Sugar Type on Ethanol Production by C. sporogenes
Research Question
What is the effect of the type of sugar molecule (glucose or cellulose) on the amount of ethanol
produced by the bacteria Clostridium sporogenes through consumption of the sugar?
Aim
The aim of this experiment is to explore the effects of different sugars on the amount of
ethanol produced by the bacterium C. sporogenes. Specifically, the goal of this experiment is to
find the differences in ethanol production when the bacterium consumes glucose instead of
cellulose, ethanol being a main waste product. Another goal is to use the results to determine if
there is a difference in efficiency in ethanol production based on sugar molecules and therefore
which sugar would be most useful in the production of ethanol for commercial purposes.
Hypothesis
It is hypothesized that the bacteria consuming glucose will produce more ethanol than
bacteria consuming cellulose or bacteria in the control environment with no added sugars. This is
due to the fact that glucose is a basic monosaccharide, while cellulose is a polysaccharide – a
much larger, complex, and more stable molecule. Cellulose, or plant fiber, is believed to be much
more difficult for the bacteria to break down, therefore being a less efficient source of food. This
also leads to the hypothesis that there is an overall difference in the efficiency of ethanol
production based on the sugar molecule used. If these hypotheses hold true, bacteria processing
cellulose would therefore yield less ethanol than those processing glucose would in the same
amount of time.
Bingham 000916-015 5
Effect of Sugar Type on Ethanol Production by C. sporogenes
Background Information
Ethanol is usually produced by organisms such as yeast and bacteria via a process called
anaerobic respiration. In general, this process consists of the sugar glucose being converted into
energy without the presence of oxygen via glycolisis, with the byproduct of lactic acid (Hine,
2005). One form of anaerobic respiration is fermentation, where the products consist of carbon
dioxide and ethanol (Hine, 2005). According to Najafpour and Lim, (2002). yeasts such as
Saccharomyces cerevisiae and S. uvarum are most commonly used for industrial production of
ethanol. However, organisms that yield the highest amounts of ethanol and are least susceptible
to ethanol inhibition are anaerobic bacteria such as Zymomonas mobilis, Clostridium indolis
(pathogenic), and C. sporogenes. Theoretically, both bacteria and yeast can produce a 98%100% yield of alcohol for any sugar or starch, however the aforementioned bacteria consistently
produce at least 92% yields compared to yields of 86% by yeast (Najafpour & Lim, 2002). The
main reason bacteria are not used despite their high ethanol production rates is due to the fact
that they typically have higher amounts of byproducts in the process such as acetic acid, and
have a harder time breaking down complex sugars than yeast do (Najafpour & Lim, 2002). In
this experiment, the bacteria with the highest expected ethanol yield, C. sporogenes, was chosen
to test if a less complex sugar allows for more efficient ethanol production, therefore possibly
making it a more likely candidate for industrial purposes.
Industrially, cellulose is the main source of sugars for ethanol fermentation because it is
cheaper than simple sugars and starchy materials that are typically used for human food instead
(Badger, 2002). Both yeasts and bacteria are being engineered to be better able to process
cellulose as efficiently as possible for industrial production of ethanol (Mackenzie, 2006).
Mackenzie (2006) listed these microorganisms as the key architects of all industrial ethanol
Bingham 000916-015 6
Effect of Sugar Type on Ethanol Production by C. sporogenes
production, and argued that both bacteria and yeasts have been engineered recently to already be
more efficient with cellulose. However, Najafpour and Lim (2002) argued that cellulosic plant
fiber is still the most difficult to break down, even if it eventually yields as much as other sugars.
The disagreement between these sources is likely a result of the difference in the times that these
sources were published. For this experiment, glucose was tested against cellulose to see if the
difference in efficiencies based on sugars does currently apply.
Regulation of the bacteria itself is extremely important to maintaining a well-designed
experiment. According to Hausner (2006), anaerobic bacteria must be maintained at a
temperature of 37° Celsius in an air-tight container. Beforehand, they must be stored in a
refrigerator to prevent growth before sub culturing. It is recommended to grow these bacteria on
a basic nutrient agar or broth. A strict maintenance and method of testing using only sterile
materials is essential, because contamination happens easily (Hausner, 2006). Much of this
experiment was based off of these rules and slightly modeled after the set up for bacterial growth
and maintenance in the experiment by Allison and Macfarlane (1990) that tested protease
production in C. sporogenes. In their experiment, the bacteria was kept in a nutrient broth and
grown in batch cultures before being moved to reaction vessels for trials. The bacteria were
maintained in an anaerobic environment with a stable temperature of 37° Celsius. This
experiment was set up in the same way, using test tubes instead of reaction vessels to fit the
purpose of the experiment.
Ethanol is typically gathered in large amounts, using systems requiring mechanisms such
as an Immobilized Cell Reactor (Najafpour & Lim, 2002). However, these systems do not work
as efficiently for such small amounts as those tested in this experiment, and are, more
importantly, typically unavailable in a classroom laboratory situation. This experiment was
Bingham 000916-015 7
Effect of Sugar Type on Ethanol Production by C. sporogenes
therefore limited and had to rely on the natural separation of ethanol from aqueous solutions.
Furthermore, for pure ethanol to be obtained, distillation is the typical method. However, it
typically results in the evaporation of a small amount of ethanol (Najafpour & Lim, 2002). This
amount is large enough to skew the data when working with such small amounts, forcing
reliance on the natural separation of ethanol from aqueous solutions in this experiment. Ethanol
can be identified on its properties as a clear, colorless liquid that is flammable when pure, with
an alcoholic odor (Najafpour & Lim, 2002). These properties helped to identify the ethanol in
this experiment.
Variables
This experiment tested amount of ethanol produced by bacteria depending on what sugar
molecule they consumed. All other variables were therefore minimized and held constant. All
equipment used was sterile to avoid contaminants. All bacteria were cultured for the same
amount of time and applied to each trial in the same amounts in an effort to keep the number of
bacteria in each trial as similar as possible. All trials used the same amount of nutrient broth.
Each trial containing sugars used one gram of the given sugar. In this way, each culture of
bacteria consuming sugar had access to more sugar than they would be able to consume in the
allotted time. This was in an attempt to keep from limiting the bacteria in how much sugar they
could consume, and therefore how much ethanol they could produce. The bacteria were therefore
able to produce as much ethanol as possible during the experiment. A set of control trials in
which no sugar was added was used to ensure that the ethanol being produced came from the
sugars tested rather than any sugars in the nutrient broth. The environment in which the bacteria
were tested was kept dark and at a constant temperature of 35° Celsius, and was sealed due to the
Bingham 000916-015 8
Effect of Sugar Type on Ethanol Production by C. sporogenes
anaerobic nature of the bacteria. All variables that may have affected the ethanol production by
the bacteria were in this way controlled.
Method
Preparation and Set Up
Before beginning the experiment, a test tube culture of C. sporogenes was grown. The
culture was allowed to grow in a sealed test tube placed inside of an incubator for three days.
The incubator was kept at a temperature of 35° Celsius to maximize growth. Because of the
small size of the tube culture, and to avoid letting the bacteria poison themselves in an excess of
waste, two tube cultures had to be grown in this fashion to have enough for all trials.
In setting up the experiment, a beaker of nutrient broth (made from distilled water and dry
nutrient mix powder) was mixed, sealed, and then put in a pressure cooker to sterilize the
solution. In making the nutrient broth, 0.8g of powder was added per 100mL of distilled water.
During this process, the pressure cooker was set at a low to medium heat setting and allowed to
reach 15 psi. The solution was then removed and allowed to cool for 15 minutes, until it returned
to room temperature. Five milliliters of the solution was then added to each of 50 large test tubes.
These test tubes were sterile, could hold ten milliliters of liquid at maximum, and had screw-on
caps to provide an anaerobic environment. In 20 test tubes, one gram of glucose was added to
each and then allowed to dissolve and mix in to the nutrient broth by inverting each test tube
gently multiple times. In 20 test tubes, the same process was done using cellulose. The remaining
10 test tubes served as control samples, and no sugar was added to the nutrient broth. Using a
sterile pipette, four drops of the bacteria tube culture was then added to each tube. The tubes
Bingham 000916-015 9
Effect of Sugar Type on Ethanol Production by C. sporogenes
were sealed and placed in the incubator, where they were kept at 35° Celsius for four days to
allow the bacteria to consume and process the sugar.
Data Collection
After four days, the test tubes were removed. Because the tests were small-scaled and
were expected to yield small amounts of ethanol per trial, the solution was transferred to smaller
test tubes where measuring liquid volumes can be more precise. Two milliliters of the solution
were removed from each large test tube by using a sterilized pipette to draw from the top of the
solution, where most of the ethanol should be. The solution was then placed into the smaller
tubes. These tubes held, at maximum, three milliliters of liquid. The solution was allowed to
settle for ten minutes, so that any ethanol would separate and rise to the top of the solution. This
separated ethanol was then measured in the test tube using a clear metric ruler. It was identified
as the clear liquid that separated on top of the solution. From the base of the meniscus of the
solution to the base of the meniscus of the ethanol, the ethanol was measured to the nearest ½
millimeter. These test tubes held one milliliter of liquid per every 15 millimeters, and this was
used to convert the measurements of each trial into milliliters. All data were recorded in a table
format.
Bingham 000916-015 10
Effect of Sugar Type on Ethanol Production by C. sporogenes
Processed Data
Table 1:
Average Amount (ml) of Ethanol Produced by C. sporogenes per 2 ml of Solution, Depending on Which
Sugar was Added to the Solution (+/-0.017), Including Standard Deviation
Type of sugar added
Average amount of ethanol
produced (ml)
Standard Deviation
Glucose
.082
.024
Cellulose
.037
.021
No added sugar
0
0
Note. All raw data and sample calculations used in conversions and processing data can
be found in Appendix A.
Bingham 000916-015 11
Effect of Sugar Type on Ethanol Production by C. sporogenes
Average Ethanol Production Based on Type of Sugar
(Error bars represent Standard Deviation)
Average Amount of Ethanol Produced (ml)
0.11
0.1
0.09
0.08
0.07
0.06
0.05
0.04
0.03
0.02
0.01
0
Glucose
Cellulose
No added
sugar
Type of Sugar Processed by C. Sporogenes
Figure 1: Average amount of ethanol (ml) produced by C. sporogenes per 2 ml of solution depending on
which sugar was consumed (+/-0.017).
Bingham 000916-015 12
Effect of Sugar Type on Ethanol Production by C. sporogenes
Data Analysis
The data shows a distinct difference between the amounts of ethanol produced by C.
sporogenes when consuming cellulose or glucose. As shown by Table 1, when consuming
glucose the bacteria produced an average of 0.082 ml per 2 ml of solution. Every individual trial
fell between 0.033 ml and 0.13 ml, with 17 out of the 20 trials reading either 0.067 or 0.10 ml. In
contrast, bacteria consuming cellulose only yielded an average of 0.037 ml per 2 ml of solution.
Each individual trial fell between 0 and 0.067 ml, with 15 out of the 20 trials reading at or below
0.033. These results show that bacteria consuming glucose produced, on average, more than
twice the amount of ethanol than bacteria consuming cellulose did. Table 1 also shows that the
bacteria in control trials, where no sugar was added, never produced any measurable amount of
ethanol. This confirms that all significant amounts of ethanol produced in the trials testing sugars
was processed by the bacteria directly from the consumption of added sugars. It was noted that a
strong alcoholic odor came from the trials along with the odor of the bacteria, helping to confirm
a significant presence of ethanol before measuring the quantitative results.
The standard deviation, as shown in Table 1, was in each case less than 0.025 ml. As
illustrated by Figure 1, the error bars representing the standard deviation of each trial do not
overlap. In the cases of the trials containing sugar, the error bars reach the same amount.
However, they also do not overlap. This shows that the ranges of error do not significantly affect
the results. The data collected in this experiment were therefore statistically significant, and the
differences in results between tested variables are conclusive data. That C. sporogenes produced
nearly twice as much ethanol when given glucose over when given cellulose is therefore a valid
result.
Bingham 000916-015 13
Effect of Sugar Type on Ethanol Production by C. sporogenes
Conclusion
The purpose of this experiment was to explore the effects of different sugars on the
ethanol production of C. sporogenes. Two sugars were tested: cellulose, a complex
polysaccharide plant fiber that is typically used in ethanol production, and glucose, a simple
monosaccharide. As per hypothesis, it was deduced that when the bacteria consume the basic
sugar, glucose, they would produce more ethanol in a set time period than when consuming
cellulose because the glucose, as a smaller molecule, would be easier for the bacteria to break
down and process. Therefore, the bacteria would be able to more efficiently consume glucose
than cellulose, leading to a higher yield of ethanol. The experiment was therefore designed to
specifically test the amount of ethanol the bacteria made in a set time based on the sugars they
consumed.
Both of the two sugars tested provided solid results. The data shows that glucose yielded
over twice as much ethanol as cellulose did, with the bacteria producing an average of 0.082 ml
compared to 0.037ml (Table 1). The data remained consistent over all trials, and the ranges of
errors, shown by standard deviation error bars, do not overlap (Figure 1). This confirms the data
as statistically significant, because it shows that even when allowing for error, the data still
suggest a difference in ethanol production. This allows for a definitive conclusion based on the
results of the experiment. It is therefore deduced that C. sporogenes produces more ethanol
when consuming glucose compared to when it consumes cellulose. This conclusion completely
supports the base hypothesis made before the experiment. It suggests that the bacteria were able
to break down and process more of the simple sugar, glucose, than the complex polysaccharide,
cellulose, leading to larger amounts of ethanol being produced by bacteria consuming glucose.
Bingham 000916-015 14
Effect of Sugar Type on Ethanol Production by C. sporogenes
This conclusion also allows speculation on another aim of this experiment. It was
tentatively hypothesized that the efficiency in ethanol production, in general, is dependent on the
type of sugar molecule used in the process. This is supported in the results because the ethanol
production in this experiment was completely dependent on what type of sugar the bacteria
consumed and processed into ethanol. Because this experiment was designed to test ethanol
production based on sugars, and the results show that the smaller sugar molecule led to more
efficient ethanol production, it is possible to infer that ethanol production depends on the type of
sugar used. However, this cannot be concluded as a certainty for all ethanol production in
general. The experiment only tested ethanol production using the bacteria C. sporogenes. There
are multiple other species of bacteria that also process sugar into ethanol, and some yeast also do
that. This experiment tested no other bacterium or yeast. Therefore, all conclusions regarding
ethanol production based on sugar molecule are based solely in context of the tested bacterium
species. Applying that conclusion to ethanol production in general can only serve as supporting
evidence for such a hypothesis that would need to be tested using several species of bacteria and
yeast.
Mentioned among the aims of this experiment was the possibility of applying the results
of this experiment to a commercial ethanol production setting. However, the experiment only
serves to provide supporting evidence to these speculations rather than proof or substantially
conclusive data. The experiment shows that C.sporogenes produces more ethanol in a set time
period when consuming glucose rather than cellulose. This allows for speculation that ethanol
production in general is more efficient when using glucose, which would mean in commercial
sense glucose would be more useful. Again, though, this can only be inferred, rather than
concluded as an absolute.
Bingham 000916-015 15
Effect of Sugar Type on Ethanol Production by C. sporogenes
In summary, the results of this experiment support the hypothesis that C. sporogenes
produces more ethanol in a set period of time when consuming glucose than it does when
consuming cellulose. This shows that the bacteria is more effective at processing glucose than
cellulose, and that ethanol production by bacteria does depend on the type of sugar used. These
findings suggest that the smaller sugar molecule, glucose, allows for more efficient ethanol
production. This supports but does not confirm the speculation that the efficiency of ethanol
production in general may depend on the sugar used, and that in a commercial sense the smaller
sugar molecules such as glucose may be the most useful.
Evaluation and Uncertainties
A reflection on the experiment reveals an evaluation of the design, results, and
uncertainties. The experiment was designed to specifically test the production of ethanol by the
bacteria C. sporogenes as being dependent on the type of sugar consumed by the bacteria. All
other variables were controlled as much as possible, and the trials lead to conclusive data. From
the results it was found that the efficiency of ethanol production is dependant of the type of sugar
consumed. However, because this experiment tested only C. sporogenes rather than multiple
species of ethanol-producing bacteria or yeast, it doesn’t allow for this conclusion to be applied
with complete certainty to all ethanol production. As far as confirming that all ethanol
production is dependent on type of sugar used in the process, this experiment is inconclusive.
Further studies of ethanol production efficiency that include testing on other ethanol-producing
agents in a fashion similar to this experiment would provide more evidence and could contribute
to a conclusion concerning all ethanol production. Furthermore, this test was designed to only
test two types of sugars, cellulose and glucose. These sugars are extremely different from each
Bingham 000916-015 16
Effect of Sugar Type on Ethanol Production by C. sporogenes
other, and so provide a good variable change for testing. However, they were the only two tested,
and it is possible that not all other sugars behave the same way. So, while the experiment did
provide data that supports the conclusion that ethanol production efficiency depends on the type
of sugar, it is not necessarily true that all large sugar molecules would have results more similar
to cellulose or that all simple sugars would have results more similar to glucose. Therefore,
further extrapolation on how the type of sugar affects ethanol production would be inconclusive
based on this data. More types of sugars would need to be tested, using the same experimental
design, to find conclusive evidence.
As for uncertainties, it is possible that flaws in the experiment could have affected the
data. All variables were controlled as well as the circumstance of a classroom environment
would allow. It is unlikely that any uncertainties would come from contamination, because all
equipment was sterile. Similarly, it is improbable that any uncertainties would have come from
uneven amounts of solution or a change in environment because those factors were also
completely controlled. However, it is possible that the number of individual bacteria might have
varied between trials. This is because the bacteria were kept in a test tube culture and transferred
via pipette. While the same number of drops of bacteria were added to each trial, it was
impossible to ensure that each drop contained exactly the same number of bacteria. So, that
could have affected the culture size in each trial. Similarly, the bacteria numbers could have been
different depending on which tube culture they came from. Because of the size of the
experiment, two tube cultures were needed to have enough bacteria. Each tube was cultured in
the exact same way, in the same environment and for the same amount of time. However, it is
still possible that there was a higher concentration of bacteria in one than in the other. Another
uncertainty comes from the fact that it was impossible to completely separate the ethanol from
Bingham 000916-015 17
Effect of Sugar Type on Ethanol Production by C. sporogenes
the solution and purify it in the experiment. Because of this, it is possible that some by-products
such as acetic acid may have been in the ethanol measured, and that not all ethanol was gathered
from every trial. Because of this, the amounts of ethanol recorded may have been slightly
different from the amounts produced in actuality. However, this error was marginal at most.
These were the main sources of uncertainty in the experiment. Even with these errors, the data is
conclusive enough to suggest that, if it was affected by differing amounts of bacteria, it wasn’t
enough to skew the data to a point where the results weren’t statistically significant. Overall, the
experimental setup and design provided clear results and a solid conclusion.
Bingham 000916-015 18
Effect of Sugar Type on Ethanol Production by C. sporogenes
References
Allison, Clive, & Macfarlane, George T. (1990). Regulation of protease production in
Clostridium sporogenes. Applied and Environmental Microbiology, 56, 3485-3490. doi:
0099-2240/90/113485-06$02.00/0
Badger, P.C. (2002). Ethanol from cellulose: A general review. In J. Janick and A. Whipkey
(eds.), Trends in new crops and new uses. (p. 17–21) ASHS Press, Alexandria, VA.
Retrieved from http://www.hort.purdue.edu/newcrop/ncnu02/v5-017.html
Hauser, Julian T. (2006). Techniques for Studying Bacteria and Fungi. Carolina Biological
Supply Company, U.S.A.
Hine, Robert (Ed). (2005). The Facts on File Dictionary of Biology (fourth edition, pp 18, 158).
New York, NY: Facts on File Science Library.
Mackenzie, Meredith. (2006). Microorganisms key to Ethanol Production. In Genetically
Engineered Microorganisms. Retrieved from http://www.nwrage.org/content/
microorganisms-key-ethanol-production
Najafpour, G.D., & Lim, J.K. (2002). Fermentation of Ethanol. In Evaluation and Isolation of
Ethanol Producer Strain SMP-6. Retrieved from http://www.andrew.
cmu.edu/user/jitkangl/Fermentation%20of%20Ethanol/Fermentation%20of%20Ethanol.h
tm
Bingham 000916-015 19
Effect of Sugar Type on Ethanol Production by C. sporogenes
Appendix A
Raw Data and Sample Calculations
Raw Data:
Table 1:
Amounts of Ethanol (as measured in mm and converted into ml) Produced per 2 ml of Solution by C.
sporogenes When Processing Glucose
Trial
Measured amount
(+/- 0.25 mm)
Measured amount
converted into ml
(+/- 0.017 ml)
1
1
0.067
2
1.5
0.10
3
1
0.067
4
1.5
0.10
5
2
0.13
6
1
0.067
7
1
0.067
8
1
0.067
9
1.5
0.10
10
0.5
0.033
11
1
0.067
12
2
0.13
13
1
0.067
14
1.5
0.10
15
1.5
0.10
16
1
0.067
17
1
0.067
18
1.5
0.10
19
1
0.067
20
1
0.067
Bingham 000916-015 20
Effect of Sugar Type on Ethanol Production by C. sporogenes
Raw Data (continued)
Table 2:
Amounts of Ethanol (as measured in mm and converted into ml) Produced per 2 ml of Solution by C.
sporogenes When Processing Cellulose
Trial
Measured amount (+/0.25 mm)
Measured amount
converted into ml (+/0.017 ml)
1
0.5
0.033
2
0
0
3
0.5
0.033
4
1
0.067
5
0.5
0.033
6
0.5
0.033
7
1
0.067
8
0
0
9
0.5
0.033
10
0.5
0.033
11
0.5
0.033
12
0.5
0.033
13
1
0.067
14
1
0.067
15
0.5
0.033
16
0.5
0.033
17
0.5
0.033
18
1
0.067
19
0
0
20
0.5
0.033
Bingham 000916-015 21
Effect of Sugar Type on Ethanol Production by C. sporogenes
Raw Data (continued)
Table 3:
Amounts of Ethanol (as measured in mm and converted into ml) Produced Per 2 ml of Solution by C.
sporogenes When in Environment With No Added Sugar
Trial
Measured amount
(+/- 0.25 mm)
Measured amount
converted into ml
(+/- 0.017 ml)
1
0
0
2
0
0
3
0
0
4
0
0
5
0
0
6
0
0
7
0
0
8
0
0
9
0
0
10
0
0
Sample Calculations:
Milliliter values were found by converting millimeters based on the number of millimeters per
milliliter in the test tubes used for recording. This conversion was 15 mm per every 1 mL:
1mL  measured mm
= mL ethanol
15mm
Ex:
1mL  1 mm
= 0.067 mL ethanol
15 mm
Averages were found by adding values of all trials from one sugar test and dividing by the
number of trials:
Bingham 000916-015 22
Effect of Sugar Type on Ethanol Production by C. sporogenes
Sample Calculations (continued):
trial 1 + trial 2 + …+ trial 20
 Average mL
20 trials
Ex:
0.067mL  0.10mL  ...  0.067mL
= 0.082mL
20
Standard deviations of the averages were found by subtracting the average amount of sugar
produced from each individual trial and square each result. Then those values were added
together. This value was divided by the number of trials. Then the square root was found,
providing the standard deviation:

1
sugar produced in trial 1 - average 2  sugar produced in trial 2 - average
20
2  ...  sugar produced in trial
= Standard Deviation
Ex:


1
0.067 - 0.0822  0.10 - 0.0822  ...  0.067 - 0.0822 = 0.24
20
20 - average 
2
