Internal Assessment: Biology SL

The Effect of Different Types of Flour on Carbon Dioxide Production and the Dough’s Growth
During Yeast Fermentation

Candidate code: kqs707

November 2023

Word count: 7400

 The Effect of Different Types of Flour on Carbon Dioxide Production and the Dough’s Growth During Yeast
(Saccharomyces cerevisiae) Fermentation

1. INTRODUCTION
The making of bread has always been fascinating to me, because of the way the dough increases its size, looking
almost like magic through the eyes of my younger self. However, as I grew older, I became interested in the art
behind its biological process, stumbling upon yeast fermentation, also known as ethanol fermentation, where
pyruvate produced through glycolysis is turned into ethanol and carbon dioxide through anaerobic respiration
(Biology Dictionary, 2019). After researching the subject I learned that yeast, being a microorganism, grew by being
supported by compounds such as sugars and proteins (Maicas, 2020), therefore mixing it with substances with these
biomolecules would target fermentation. Hence, I decided to try it, noticing that the carbon dioxide produced in this
process caused the substance to grow. Therefore, I experimented with different flours to see if yeast reacted distinctly
with this variety of options to determine if the different amounts of organic molecules stored in each flour would
affect its height after fermenting, to analyze the effect of cell respiration on the production of carbon dioxide and,
hence, the dough’s growth.
When thinking about bread, most people believe that the only option available is made with wheat flour, however,
nowadays many other opportunities to follow a more beneficial diet have brought alternatives to this popular choice.
Thus, multiple seeds, grains, and even some fruits, have been discovered to make a very similar, and sometimes
more beneficial option of bread. For people who are not able to consume it because of different health conditions,
like celiac disease, or those who are trying to achieve a more varied diet, these new alternatives can be life-changing
and enriching for their bodies. Therefore, I have meticulously selected a variety of flours, to determine the different
options available to make a different type of bread that can help achieve a more appropriate diet for everyone. Hence,
the objective of this investigation is to determine the type of flour in the fermentation process of yeast, in this case
using the Saccharomyces cerevisiae species, to produce more carbon dioxide by measuring the height of the
substance by analyzing the percentage of variation of these two dependent variables.
2. EXPLORATION
2.1 Background Information
What is cell respiration?
Cell respiration is a process in which cells turn sugars into energy through the ATP molecule to powder cellular
reactions. To create ATP, cells need fuel and an electron acceptor that conducts the chemical process and turns the
energy into a usable form. There are two types of cellular respiration: aerobic and anaerobic. Aerobic respiration
requires oxygen (powerful electron acceptor) and is an efficient process that allows eukaryotes to make complicated
functions and have active lifestyles, hence requiring a constant supply of oxygen. In this type of respiration, glucose
is combined with oxygen and ADP (ADP + Pi = ATP), producing 𝐶𝑂2, water, and ATP. Once the carbon molecules
of glucose are entirely broken down, they are exhaled as 6 molecules of carbon dioxide. The other type of cellular
respiration is anaerobic respiration, a process that produces small amounts of ATP without the presence of oxygen.
Alcoholic fermentation and Lactic acid fermentation are two types of anaerobic respiration. The first one, performed
by yeasts and smaller cells, metabolizes sugar to produce alcohol and carbon dioxide as byproducts. The second one,
performed by human muscle cells and some bacteria, is used to produce lactic acid, by breaking one molecule of
glucose into two molecules of this acid (Buckley, 2021).
Aerobic respiration formula:
𝐶6𝐻1206 (glucose)+ 6 𝑂2 + 36 ADP (depleted ATP) + 36 Pi (phosphate groups)→ 6 𝐶𝑂2 + 6 𝐻2𝑂 + 36 ATP

What is fermentation?
Fermentation is a metabolic process where organic molecules, normally glucose, turn into acids, gases, or alcohol in
the absence of oxygen or electron transport chains. Its pathways regenerate nicotinamide adenine dinucleotide
coenzymes (NAD+) used in glycolysis to release energy as Adenosine triphosphate (ATP), only yielding two of them
per glucose molecule through the process of glycolysis (Biology Dictionary, 2019).
This process occurs in certain types of bacteria and fungi, which live in an oxygen-free environment in order to
survive (obligate anaerobes), in facultative anaerobes like yeast (unicellular fungus), and in muscle cells when there

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is a short oxygen supply. It is important for food and beverage industries, converting sugars into ethanol used to
produce alcoholic drinks, the release of 𝐶𝑂2 yeast in bread making, and the production of organic acids to flavor and
preserves dairy products and vegetables. The main function of fermentation is converting NADH back to NAD+
coenzyme to be used in glycolysis. During the process, an organic electron acceptor, like pyruvate or acetaldehyde,
reacts with NADH to form NAD+, producing carbon dioxide and ethanol, known as ethanol fermentation, or lactate
for lactic acid fermentation (Biology Dictionary, 2019).
Alcohol fermentation formula:
𝐶6𝐻1206 (glucose) + 2 ADP (depleted ATP) + 2 Pi (phosphate groups)→ 2 𝐶2𝐻5𝑂𝐻 (ethyl alcohol) + 2 𝐶𝑂2 + 2 ATP

Yeast and its characteristics

Yeast is a eukaryotic microorganism that lives mainly in water, soil, air, or plant and fruit surfaces, directly
intervening in ripe fruit decomposition and participating in the fermentation process. This organism can carry out its
metabolic and fermentation activity since it has the necessary nutrients and substrates. Its growth is supported by
essential compounds like sugars, amino acids, vitamins, minerals, and oxygen. They present a high morphological
divergence (round, ellipsoidal, and oval-shaped) and reproduce mainly by budding, asexual reproduction, resulting in
a new and genetically identical cell, preserving the genotype, and maintaining a stable fermentation behavior.
Additionally, they tolerate acidic environments (pH 3.5 or less) and are capable of fermenting a high spectrum of
sugars, like glucose, fructose, maltose, sucrose, and maltotriose predominate (Maicas, 2020).
Yeasts are divided into two groups: Saccharomyces and non-Saccharomyces. The first group can be round or
ellipsoidal and is the most used in wine and beer fermentation because of its fermentative capacity: rapid growth and
easy adaptation. They are able to tolerate concentrations of 𝑆𝑂2 that most non-Saccharomyces yeasts don’t survive.
The most studied species is Saccharomyces cerevisiae (S. cerevisiae). However, this second group has high
metabolic differences that allow them to work effectively in fermentation processes, because they allow the synthesis
of several products. Even though they have lower fermentative power and production of ethanol, they possess the
ability to produce high levels of aromatic compounds like fatty acids. Moreover, the fermentative activity of this
group of yeast occurs in the presence of little oxygen, leading to an increase in cell biomass and a decrease in ethanol
yield (Maicas, 2020).
Ethanol fermentation, which is typically performed by yeast, is a process where pyruvate, which has been produced
through glycolysis, turns into ethanol and 𝐶𝑂2 in two steps: firstly, the pyruvate releases 𝐶𝑂2 to form a two-carbon
compound (acetaldehyde), and later it is reduced by NADH to ethanol, generating the NAD+ to be used in
glycolysis. This way, the glucose molecule is transformed into two 𝐶𝑂2 molecules and two ethanol molecules
(Biology Dictionary, 2019). Furthermore, yeast fermentation requires the right temperature for it to thrive, or else the
yeast will be killed in the wrong conditions. The ideal temperature when utilizing dry yeast is between 100° and
110°F (38°-43°C) to achieve the best results in the fermentation reaction (Fleischmann’s Yeast, n.d.).
Formula: GLUCOSE → 2 ETHANOL + 2 CARBON DIOXIDE
𝐶6𝐻12𝑂6 → 2 𝐶2𝐻5𝑂𝐻 + 2 𝐶𝑂2

What is flour?
Flour is a powdery substance made of pulverized dry grains through a milling process. Any grain can be turned into
flour, however, the most common one is wheat. Apart from the type of grain, flour varies depending on the part of
the grain it contains, which can be endosperm, bran, or germ. Endosperm is the starchy center of the grain,
containing carbohydrates, proteins, and small amounts of oil (lipids). Gluten is a protein found in this part of the
grain, giving strength, elasticity, and a chewy texture to pasta, bread, and pizza dough. On the other hand, bran is the
outer husk of the grain, adding texture, color, and fiber to the flour. Germ, the reproductive epicenter of the grain, is
the source of nutrients (the flour that retains germ in the milling process has more fiber, minerals, and vitamins)
(Moncel, 2023).
Wheat flour has large amounts of starch (Sindhu et al., 2021), conforming 90% of the total carbohydrates that
contain it (Arnason, 2019). Moreover, it contains gluten proteins, which interact with hydrogen and hydrophilic
bonds, creating a viscoelastic dough. It’s also able to retain gas produced by cellular respiration (Hoseney, Rogers,
1990). Furthermore, rye flour has high amounts of carbohydrates, but it has slightly less of them than wheat flour. It

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contains gluten as well, however, it has less glutenin, which is important for forming strong gluten bonds that are
necessary for creating a stretchy dough (Donia, 2022). Additionally, almond flour has low quantities of
carbohydrates and is gluten-free, so it does not build a thick and gluey consistency in the dough (Mikstas, 2022). On
the other hand, coconut flour has a lower glycemic index than other flours like the wheat alternative, making the
carbohydrates take longer to be digested and absorbed. Adding to this, it is gluten-free too (Mikstas, 2023). Talking
about flax seeds, they are made up of 29% carbohydrates, however, 95% of them are fiber, meaning that the amount
of digestible carbohydrates in flaxseed flour is low (Bjarnadottir, 2019). In relation to sesame flour, it also has low
amounts of carbohydrates, containing around 34 grams of it per cup (in most variants) and it’s also known to be
gluten-free (Clockwork Lemon, n.d.).
How does yeast fermentation with flour work?
Yeasts feed on the sugars that come from the complex starch molecules (complex carbohydrates) that flour contains.
These molecules are broken down into simpler sugars by enzymes, and catalysts, inside the flour when it is hydrated,
making them accessible to yeast, since they cannot break down starch by its lack of amylase. Yeasts, therefore, break
down simple sugars, like glucose and fructose, into smaller and simpler molecules for energy from which it grows
and multiplies by mitosis and lets out a liquid that releases ethanol and carbon dioxide into air bubbles in the dough
(Phillips, 2000).
2.2 Research Question and Objectives
Research question:
What type of flour (wheat, rye, almond, coconut, sesame, and flaxseed) causes yeast (S. cerevisiae) fermentation to
produce more carbon dioxide (ppm), hence increasing the substance’s height (cm) (determined by inserting a mixing
rod in the center of the dough until it touches the bottom of the beaker and measuring it with a vernier digital ruler)?
Objectives:
● Determine the type of flour in the fermentation process to produce more carbon dioxide by measuring the
height of the substance.
● Determine what type of flour is more efficient for cell respiration.
2.3 Hypothesis
The wheat flour will make the yeast (S. cerevisiae) fermentation reaction produce more carbon dioxide (ppm), thus
making the substance increase more in height (cm), because it has the maximum amounts of carbohydrates, which
means that it has more glucose to produce cell respiration, therefore, producing more amounts of carbon dioxide.
2.4 Variables
Table 1. Independent and dependent variables, including measuring ways and instruments
MEASURING
VARIABLE VARIABLE NAME RANGE / MEASURING WAYS INSTRUMENTS/UNITS/
UNCERTAINTY
Type of flour Ranges: flaxseed, almond, wheat, Beakers with labels to
Independent
coconut, sesame, and rye flour identify which is which
Amount of carbon dioxide height - centimeters (cm) (in %) digital vernier ruler
Dependent produced variation in the concentration of (±0.001cm), digital carbon
Height increase of the dough carbon dioxide - ppm (in %) dioxide vernier sensor (±10%)

Table 2. Controlled variables, why they should be controlled, and how to control them

VARIABLE WHY CONTROL IT? HOW TO CONTROL IT

Amount of Using too much or too little yeast may affect the reaction since A scale to weigh the amount of
yeast in each it needs to be quantified so the fermentation works (avoiding yeast used for each beaker
beaker lack of flour or water for fermentation to happen or stopping (Make sure the yeast weight is
the reaction for having too little product for the amount of the same in each beaker)
(2 grams of yeast per beaker)

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 flour). Also, the different quantities in each beaker affect the
reactions differently because of the different quantities used.
Amount of Using too much or too little amounts of flour may affect the A scale to weigh the amount of
flour per reaction since it needs to be quantified so the fermentation flour used for every beaker (the
beaker works (avoiding lack of yeast or water for fermentation to flour in each beaker must weigh
happen or stopping the reaction for having too little product for the same)
the amount of yeast). Also, using different amounts of flour (30 grams of flour per beaker)
wouldn’t allow me to test correctly the different ways yeast
interacts with each type of flour, since the different quantities
would change the results.
Amount of The reaction occurs efficiently during a certain period of time. A chronometer to record the
time to record After that, the dough starts decreasing in size and, therefore, time that has passed, setting it to
data losing its height. Because of this, it is necessary to establish a go backward for 20 minutes.
small period of time, so the reaction can happen as long as
possible without it reversing its effects and altering the results.
Temperature of The temperature of the water affects the effectivity the yeast A thermometer for the water, to
the water will work with, so the temperature needs to be regulated so it make sure it is the same for each
doesn’t affect the experiment negatively, as yeast has an beaker (so the reaction is not
optimal temperature in which it tends to work best. Also, all affected by different water
beakers will need the same temperature of the water so the temperatures)
yeast’s conditions are the same when reacting with the flour, (65°C)
making the experiment fair.
Temperature of The temperature of the room affects the temperature of the Putting all the beakers (with the
the room substance involved in the experiment, so, if it is too low or too different flours in the same
high, it may affect the yeast’s effectiveness. Also, all the room at the same time) so all are
beakers need to be at the same room temperature so the exposed to the same room
reaction in each beaker happens at the same conditions (or else conditions
the results will vary depending on the different temperatures). Using an electric radiator heater
to increase the room temperature
It is important to note that yeast fermentation is best at slightly
(28°C)
high temperatures

2.5 Materials - per trial

Material Amount Uncertainty Material Amount Uncertainty
Digital scale 1 ±0.01g Water (65°C) 400 ml -
Watch glass 6 - Chronometer 6 -
Kettle 1 - Metallic spoon 1 -
Thermometer 7 ±0.05°C 1 ½ measuring cup 1 -
Beaker (1L) 1 ±0.5% Mixing glass rod 6 -
Yeast 12g - Digital vernier ruler 1 ±0.001cm
Flaxseed flour 30g - Digital 𝐶𝑂2 vernier sensor 6 ±10%

Rye flour 30g - Recycled 4L plastic bottles 6 -

Almond flour 30g - Masking tape 1 -
Coconut flour 30g - Electric radiator heater 1 -
Wheat flour 30g - Indelible pen 1 -
Sesame flour 30g - recycled polystyrene squares 6 -
Beaker (250ml) 6 ±5% vernier screen monitor 2 -

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Graduated cylinder (50ml) 6 ±0.5ml

2.6 Procedure
Preparing the material:
1. Cut the 6 4L plastic bottles in half and put a small square of recycled polystyrene inside to create a flat surface
for each bottle.
2. Insert the 6 digital vernier 𝐶𝑂2 sensors on the mouth of each bottle and seal them with masking tape.
3. Connect the sensors to the vernier screen monitors, so they can record data.
Making the experiment
4. Grab 6 beakers (250ml) and label them from 1 to 6 with an indelible pen.
1. Put one of the beakers on the digital scale, calibrate it, and weigh 30g of wheat flour (repeat this process with
the other 5 beakers).
2. Add water to a kettle and wait for it to boil.
3. Pour the hot water into a beaker (1L) and put the thermometer on it, then add cold water until the temperature
reaches 65°C.
4. Pour 30 ml of water into a graduated cylinder (50 ml) and repeat this process for the other 5 graduated cylinders.
5. Pour the water of each graduated cylinder into each of the 6 beakers and mix it with the flour until it forms an
even dough (use a mixing glass rod).
6. Weight 2g of yeast on the digital scale using a watch glass (repeat the process 5 more times).
7. Set the chronometer to run backward for 20 minutes and add the 2g of yeast into one beaker, mix it with the rod,
and start the chronometer (repeat this process with the other 5 beakers)
8. Insert the clean side of the mixing rod in the center of the dough until it touches the bottom of the beaker, pull it
out, and leave it aside (repeat the process for the other 5 beakers).
9. Put each beaker inside the lower part of the 4L plastic bottles and put the upper part of the bottle on top (which
contains the sensor), sealing them together with the masking tape).
10. Press “record” [data] on the vernier screen monitors when the first five minutes in the chronometer have passed,
and record the first data of carbon dioxide produced by the reaction of each beaker (add it to the data table).
11.Leave the reaction to occur in a room with a 28°C temperature achieved by the electric radiator heater.
12. Return to the mixing rods that were previously left aside (on step 8) and measure the height of the dough
remaining on the rods’ side that was introduced into the mixture of each beaker, using the digital vernier ruler
(record the data and add it to the table as “initial height”, then clean the rods).
13. After 10 minutes have passed since the initial reaction (the chronometer should say 10 minutes remaining),
record the carbon dioxide produced from every beaker.
14. After 20 minutes have passed since the initial reaction (the chronometer should have stopped), record the final
carbon dioxide produced from each beaker.
15. Take the masking tape off the plastic bottles and take out the beakers from the inside.
16. Insert the mixing glass rod in the center of the dough until it touches the bottom of the beaker and pull it out,
then measure the height of the dough remaining on the rod with the digital vernier ruler (record the data in the
table and repeat this process with the other 5 beakers).
17. Wash the beakers and dry them, then repeat the process with four of the beakers until the ten trials are complete.
18. Repeat the same process with the other five flours (change the amount of water depending on the flour:
almond - 50ml, ryle - 40ml, sesame - 50ml, coconut - 150ml, and flaxseed - 80ml)
Processing the data
19.Subtract the final height from the initial height of each dough to find the height variation.
20. Find the percentage of variation in relation to the amount of water to make the results more fair and precise.
21. Subtract the final amount of carbon dioxide produced from the initial amount to find the variation from 5 to 20
minutes of reaction.
22. Find the percentage of variation in relation to the amount of water to make the results more fair and precise.
2.7 Bioethical considerations and safety measurement.
Because of security measurements, a lab coat must be used to avoid any contact between the materials and the skin
as a precaution, not only because of the hot temperatures involved in the experiment (65°) but also to prevent
transferring any contaminants that could be carried on personal clothing to the experiment materials, which could
affect the results later on. Equipment must be washed before and after the experiment so residues do not affect the

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results. It is necessary to be careful when managing boiling water, avoiding direct contact with it, by grabbing the
kettle by the handle and avoiding touching the hot beakers.
Regarding bioethical considerations, it is important to separate the exact amount of material necessary for the
experiment to prevent wasting biological material, like yeast or any kind of flour. Disconnect any electronic items
when they are not in use, to reduce the use of electricity. Clean the working environment after finishing the
experiment to allow others to use the space afterward. Additionally, the fermented dough will be used for the
making of bread after the investigation is concluded to not waste any of the material that can still have further
purposes like this one.
3. ANALYSIS
3.1 Results
Qualitative Data
Firstly, during the process of fermentation, the dough released bubbles in the reaction with every type of flour,
which started to appear after the first 5 minutes since the yeast made contact with the flour. Furthermore, for the rye
flour samples, the dough growth happened faster than the rest of the flour samples. Moreover, compared to the rest
of the flour samples, the flaxseed flour dough formed a more condensed and viscous texture, not allowing the
complete blend of the yeast with the dough.
Table 4. Raw data of carbon dioxide released from initial time (5 min from the start of
reaction) to final time (20 minutes from the start of reaction)
Carbon Dioxide Release (ppm)

Initial Carbon Dioxide release (5min) Final Carbon Dioxide Release (20min)

Almond Wheat Rye Sesame Coconut Flaxseed Almond Wheat Rye Sesame Coconut Flaxseed

Trial 1 634 270 913 1060 825 564 38705 6749 6562 3097 59474 2500

Trial 2 819 237 707 684 503 920 27671 3901 8042 2381 28959 4004

Trial 3 588 207 527 535 583 716 24678 3211 3264 1864 19399 2446

Trial 4 569 915 1475 899 878 812 20806 9528 8897 2894 51883 3865

Trial 5 894 903 512 627 625 926 35900 4981 4167 2265 28909 5008

Trial 6 678 586 625 676 1019 615 24543 2639 2650 2509 51972 3509

Trial 7 623 571 1218 911 897 525 20209 2900 6124 3169 39700 2904

Trial 8 1332 760 548 615 703 823 47959 2910 5864 2210 40271 3514

Trial 9 598 714 936 737 764 714 32451 3810 5113 2337 78229 2574

Trial 10 688 642 512 586 1025 760 30576 7403 5948 2603 68315 2730

Table 5. Raw data of the height increase from the initial (5 min from the start of reaction) to
the final time (20 minutes from the start of reaction)
Height (mm)

Almond Wheat Rye Sesame Coconut Flaxseed

Initial Final Initial Final Initial Final Initial Final Initial Final Initial Final

Trial 1 20,09 37,61 16,76 28,35 17,51 44,58 23,70 37,1 49,55 53,23 36,75 43,24

Trial 2 19,75 36,49 14,12 34,31 18,14 34,85 23,93 37,68 52,36 55,85 40,53 54,54

Trial 3 21,91 42,70 14,88 32,59 16,09 47,01 22,84 39,04 49,5 57,49 33,25 46,05

Trial 4 21,19 37,13 13,19 31,19 18,77 35,89 24,25 40,36 48,58 54,61 35,44 50,06

Trial 5 22,37 34,28 17,61 29,45 15,41 42,23 23,01 38,03 48,83 71,18 38,58 45,85

Trial 6 20,5 33,96 15,96 34,93 16,05 37,78 24,77 33,07 51,12 57,71 35,03 45,00

Trial7 22,51 32,73 20,67 32,58 16,44 40,99 24,83 31,54 46,00 62,05 35,67 46,41

Trial 8 19,66 37,41 15,83 36,03 17,38 40,17 23,37 40,62 44,80 64,34 31,27 45,52

Trial 9 24,14 36,97 19,51 30,13 19,24 47,50 23,67 30,37 49,53 54,47 33,7 45,90

Trial 10 23,71 40,16 18,52 26,86 18,29 44,22 22,99 32,51 47,16 64,45 33,35 45,99

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Calculations (all calculations will be exemplified by trial 1 of almond flour.)

To measure the height variation in the dough during yeast fermentation, it is

necessary to subtract the initial height from the final height (in cm) to find 37. 61𝑐𝑚 − 20. 09𝑐𝑚 = 17. 52𝑐𝑚
the overall growth

Since the experiment is standardized to have different amounts of flour in 𝑜𝑣𝑒𝑟𝑎𝑙𝑙 𝑔𝑟𝑜𝑤𝑡ℎ (𝑐𝑚)
× 100 = %
𝑎𝑚𝑜𝑢𝑛𝑡 𝑜𝑓 𝑤𝑎𝑡𝑒𝑟 (𝑚𝑙)
order for yeast fermentation to be efficient with every type of flour, it is
necessary to change the results to percentages in relation to the amount of 17.52
× 100 = 35. 04%
50
water used for each flour. In the case of almond flour, 50ml of water was
added.

In regards to the overall amount of carbon dioxide produced, the same

education is undertaken, subtraction the initial 𝐶𝑂2production for the final 38705𝑝𝑝𝑚 − 634𝑝𝑝𝑚 = 38071𝑝𝑝𝑚

𝐶𝑂2 production (in ppm).

Then we find the percentage of carbon dioxide produced in relation to the 𝑜𝑣𝑒𝑟𝑎𝑙𝑙 𝐶02 𝑝𝑟𝑜𝑑𝑢𝑐𝑡𝑖𝑜𝑛 (𝑝𝑝𝑚)
× 100 = %
𝑎𝑚𝑜𝑢𝑛𝑡 𝑜𝑓 𝑤𝑎𝑡𝑒𝑟 (𝑚𝑙)
amount of water added to the almond flour.
38071
50
× 100 = 76142%

The following formulas show how to find the average and standard deviation average: 𝑥̄ =
Σ𝑥
𝑁
for both the overall height and 𝐶𝑂2 production with each flour.
2
Σ(𝑥𝑖−µ)
standard deviation: σ = 𝑁

Table 7. Percentage of variation in 𝐶𝑂2 produced from the initial time to final time
Total Carbon Dioxide Release (%)(from 5min to 20min)

Trials Almond Wheat Rye Sesame Coconut Flaxseed

1 76.142,00 21.596,67 14.122,50 4.074,00 39.099,33 2.420,00

2 53.704,00 12.213,33 18.337,50 3.394,00 18.970,67 3.855,00

3 48.180,00 10.013,33 6.842,50 2.658,00 12.544,00 2.162,50

4 40.474,00 28.710,00 18.555,00 3.990,00 34.003,33 3.816,25

5 70.012,00 13.593,33 9.137,50 3.276,00 18.856,00 5.102,50

6 47.730,00 6.843,33 5.062,50 3.666,00 33.968,67 3.617,50

7 39.172,00 7.763,33 12.265,00 4.516,00 25.868,67 2.973,75

8 93.254,00 7.166,67 13.290,00 3.190,00 26.378,67 3.363,75

9 63.706,00 10.320,00 10.442,50 3.200,00 51.643,33 2.325,00

10 59.776,00 22.536,67 13.590,00 4.034,00 44.860,00 2.462,50

Average 59.215,00 14.075,67 12.164,50 3.599,80 30.619,27 3.209,88

St. deviation 17.059,47 7.575,64 4.434,43 554,92 12.386,02 923,40

Table 6. Percentage of variation in the growth of the dough after 15 minutes of reaction
Dough Growth (%)

Trial number Almond Wheat Rye Sesame Coconut Flaxseed

1 35,04 38,63 67,68 26,80 2,45 8,11

2 33,48 67,30 41,78 27,50 2,33 17,51

3 41,58 59,03 77,30 32,40 5,33 16,00

4 31,88 60,00 42,80 32,22 4,02 18,28

5 23,82 39,47 67,05 30,04 14,90 9,09

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 6 26,92 63,23 54,33 16,60 4,39 12,46

7 20,44 39,70 61,38 13,42 10,70 13,43

8 35,50 67,33 56,98 34,50 13,03 17,81

9 25,66 35,40 70,65 13,40 3,29 15,25

10 32,90 27,80 64,83 19,04 11,53 15,80

Average 30,72 49,79 60,48 24,59 7,20 14,37

St. deviation 6,38 14,94 11,61 8,20 4,80 3,56

Figure 1 shows that the rye flour was the one to increase more in height, with a percentage variation of 60.48%.
Then follows wheat flour, with a 49.79% height variation. Almond flour has a result of 30.72%, while sesame has
24.59%. Flaxseed flour increased 14.37% and coconut flour, whose height percentage variation was the lowest, has
a result of 7.20%. On the other hand, figure 2 shows that almond flour was the one with a higher percentage
variation in the carbon dioxide produced, with a result of 59,215%. Then, the next one to have a high percentage is
coconut flour, with a variation of 30,619.27%. Wheat flour’s result was 14,075.67%, and slightly lower was rye
flour, with 12,164.50%. After, comes sesame flour with a carbon dioxide production variation of 3,209.88% and the
lowest result was flaxseed flour with a result of 3,208.88%.
Analysis of ANOVA single-factor
The ANOVA single-factor test was conducted to analyze if there exists a significant difference between the six
groups of flours (categorical variables). If the significance level of the P-value is over 5% (>0.05), it shows no
significant difference. However, if the P-value is under 5% (<0.05), there is a significant difference between groups,
and therefore, the null hypothesis is rejected.
Carbon dioxide production
Null Hypothesis (𝐻0): The type of flour has no significant effect on the production of 𝐶𝑂2 during yeast
fermentation.
Alternative Hypothesis (𝐻1): The type of flour does have a significant effect on the production of
carbon dioxide during yeast fermentation.
Height increase of dough
Null Hypothesis (𝐻0): The type of flour has no significant effect on the dough growth during yeast
fermentation.
Alternative Hypothesis (𝐻1): The type of flour does have a significant effect on the dough growth
during yeast fermentation.
Table 8: Analysis of variance of the type of flour and carbon dioxide produced

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 Table 9: Analysis of variance of the dough growth

Tables 8 and 9 demonstrate that the P-value is under 0.001, which means that its significance level is under 5%
(<0.05). This shows a significant difference between the type of flour, thus both null hypotheses are rejected and
the alternative hypotheses are accepted, stating that the type of flour does have a significant effect on the dough
growth and, also on the production of carbon dioxide. Additionally, in Table 8, the value of F (52.7285475) is
bigger than F crit (2,28606986), suggesting that there is a bigger difference between groups than within groups.
Similarly, in Table 9, the value of F (50.3801964) is bigger than Fcrit (2,386969853), which also shows a bigger
difference between groups than within groups, suggesting that there is a statistically significant difference, in both
cases, in the results of my report.
3.2 Discussion
In regards to Figure 1, it is demonstrated that the height variation is the biggest when yeast reacts with the rye flour,
with a percentage of 60.48%, while the dough growth is the smallest in the reaction with coconut flour, with a result
of 7.20%. The results in this graph can be explained by different reasons. In the first place, rye contains pentosans,
which are a type of polysaccharide made up of pentoses. These biomolecules, when hydrated, create a glue-like
texture in the dough, making it sticky (Vindi, 2020). Because of the viscosity of the dough created by the pentosans,
the carbon dioxide produced through yeast fermentation is retained, which causes the expansion of the dough due to
the gases stuck in it, which elevate the substance. Additionally, this grain contains high levels of alpha-amylase
activity (Stępniewska et al., 2021). Therefore, this enzyme catalyzes the breakdown of starch molecules
(carbohydrates used by organisms to do cell respiration) into small and simple sugars (glucose). Hence, rye flour
speeds up yeast activity (Vindi, 2020). This causes fermentation rates to be faster, releasing carbon dioxide in a
shorter period of time. Because of this, more gas is trapped in the dough, increasing its height.
In contrast with the rye flour’s results, the little growth of the coconut flour fermentation can be explained by
different factors. The first one would be the lack of gluten, as it is known to be gluten-free (Mikstas, 2023). Gluten
is the name provided for proteins (commonly found in wheat, rye, barley, and triticale) that help maintain food in
shape, holding it together (Celiac Disease Foundation, n.d.). Therefore, gluten provides a strong structure to
different types of flour, which is something that coconut flour does not have. Because of the lack of these proteins,
the dough is too weak to maintain the increasing structure provided by the production of carbon dioxide. Because
of this, it limits the elasticity, and thus, the dough growth. Another factor that might have contributed to these
results is the high amounts of water combined with the coconut flour. Even though the percentages are done in
relation to the amount of water provided, it does not erase the fact that the water affected the results. Too much
water combined with the lack of gluten to strengthen the dough would not be able to hold its new structure.
The rest of the flours have a range of height variations that do not reach the higher levels of rye flour fermentation
or lower levels of coconut flour. Analyzing as an example the wheat flour, it could have grown less than rye flour
due to its different components. Even though wheat flour has high levels of gluten that allow it to maintain its
structure, the pentosans, which are barely present in wheat (Wang, 2003), helped the reaction of rye flour
fermentation, trapping the gas that increases the size, and its levels of gluten (even though smaller than wheat)
might have been strong enough to keep this new height.
Furthermore, Figure 2 shows that the highest carbon dioxide production was in yeast fermentation with almond
flour, with a variation of 59,215%, while the lowest 𝐶𝑂2 production was in flaxseed flour, with 3,209.88%. The
results that show almond flour as the highest producer of carbon dioxide cannot be fully sustained, as facts go
against this happening, because of one main reason. This is given because almonds provide around 6.1 grams of
carbohydrates, meaning that they have a low glycemic index, which shows that they have small amounts of
carbohydrates (Cervoni, 2022). This suggests that almond flour should not produce high amounts of carbon

9
dioxide, since yeast, during fermentation, does not have a large amount of glucose available to do cell respiration.
Thus, the yeast cells should be producing a lot less 𝐶𝑂2 than the results show. However, a recent study suggests that
almond has a strong effect on the TCA cycle (Krebs cycle), which is a metabolic pathway where byproducts of
nutrient digestion converge in order to produce energy. This is proposed to be because of the monounsaturated and
polyunsaturated fatty acids present in the almond group, suggesting that the increased amounts of unsaturated fatty
acids available may increase the substrates for this cycle’s activity, feeding it (Dhillon, 2021). As the process of the
Krebs cycle releases carbon dioxide, it could mean that the lipids in almond flour may be the cause of this high 𝐶𝑂2
production. Additionally, the same study demonstrates consumption increases the levels of acetylcarnitine, which is
involved in the movement of acetyl-coA, a co-enzyme that releases carbon dioxide, into the mitochondria during
beta-oxidation (Dhillon, 2021). Because of this, yeast consumption of almond flour can have stimulated the
production of this gas.
On the other hand, flaxseed flour was the one that produced less carbon dioxide during yeast fermentation because
this seed is made up mainly of fiber. Although it contains up to 29% of carbohydrates, 95% of carbohydrate it
contains is fiber, meaning that it is extremely low in net digestible carbs (Bjarnadottir, 2019). Of the fiber it
contains, two-thirds of it is insoluble, which consists of cellulose, hemicellulose, and lignin (Kajla et al., 2014).
Cellulose is made of glucose monomers packed tightly as extended long chains, linked by β 1-4 glycosidic bonds,
giving it a high tensile strength and rigidity (Lumen Learning, n.d.). Because of this, insoluble fibers like cellulose
are difficult to break down, which means that it takes the yeast longer to digest than simple carbohydrates. Since
flaxseed flour only contains a few percent of carbohydrates easily digestible by yeast, in the small period of time of
fifteen minutes of reaction, yeast will first digest this limited amount of carbohydrates which are smaller and easier
to break down to do cell respiration, and the rest 95% of fiber will take a lot longer to digest, and, although it is not
the human body, and yeast has the ability to “stimulate the growth of fiber-digesting bacteria” (Su, 2022), it will
only be able to digest a few amounts of fiber in this period of time. Because of this, it is possible that it produced
less amount of carbon dioxide because of the limited time of reaction in relation to the amount of complex
carbohydrates flaxseed has, making them impossible to digest in only fifteen minutes.
4. EVALUATION
4.1 Conclusion
In conclusion, the hypothesis that stated that the rye flour would make the fermentation reaction produce more
carbon dioxide, thus making the substance increase more in size because it has the maximum amount of
carbohydrates that would allow it to produce more amounts of carbon dioxide by cell respiration, is not fully
rejected, although it differs from the results obtained, as it is demonstrated that rye flour increases more in height
than the rest of flours during yeast (S. cerevisiae) fermentation. However, different results for each dependent
variable show that they are not strongly related to one another, rejecting the possibility that carbon dioxide will
always affect the height increase of the dough. Based on these results, it is determined that the type of flour that
produces higher levels of carbon dioxide is almond flour. This is suggested to be because of its role in the Krebs
cycle in regard to fatty acids and increased acetylcarnitine. Nevertheless, the one that increases more in size,
although producing low levels of 𝐶𝑂2 is rye flour, due to different factors like the presence of complex
carbohydrates and enzyme activity in the reaction.
4.2 Project’s strong points
Firstly, after making multiple preliminary trials, and testing many different flours, I realized that fermentation of
each flour required different amounts of water for the reaction to happen, or else the reaction wouldn’t take place
properly. Hence, I standardized the measurements for everything to work smoothly and allow the yeast to do cell
respiration correctly. Moreover, by repeating the experiment after standardizing the measurements, I noticed that
leaving the dough to react for a long period of time was making it decrease its height, which meant that the results
of the reaction were already getting altered by the factor of time. Therefore, I standardized the time to record the
data. Because of this, I set 20 minutes of reaction so the yeast would have enough time to do cell respiration
without the dough losing its height. Additionally, using more precise equipment, like the vernier equipment to
measure the carbon dioxide production and the height of the dough, I made sure the results were measured in a
more accurate way, reducing the levels of uncertainty and systematic error. Also, it was beneficial to the report to
transform the results into percentages, as it made the data a lot more fair and precise since it is delimited to the
amounts of water used to eliminate as much unfair variation between groups that existed by adding diverse
quantities of water to the flour. Finally, the investigation is enhanced by the initiative of creating a recycled

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experimental design, since it reduces the environmental damage plastic materials could bring if not reused.
Therefore, this report is contributing to reducing levels of pollution and helping climate change through an
innovative design of plastic bottles with recycled polystyrene to control the 𝐶𝑂2 produced by the reaction.
4.3 Project’s Limitations
In the first place, as my experiment was conducted in two different spaces inside the laboratory (the laboratory
itself and the storage room behind it, which had a higher temperature set for the reaction), the timeframe of
transportation of the samples could have reduced the level of accuracy due to the temperature changes, even though
the reaction is monitored after the first five minutes to reduce this possible error. A solution to this limitation would
be to conduct the experiment in one single space, which already has the optimum temperature where the yeast
reaction can thrive, which will create a better-controlled environment so the reaction is not affected by changes in
temperature.
Furthermore, The number of trials the experiment was repeated was only 10, meaning that the results could be more
accurate if they were conducted more times. Therefore, a way to solve this would be to increase the number of trials
to make sure the results are as accurate as possible to reduce the levels of uncertainty so that accepting or rejecting
the hypothesis can be well sustained. In addition, it will also make it possible to eliminate any outliers to make sure
the data is more precise. Additionally, for the experiment, different brands of flour were used due to a lack of
variety from just one brand. This means that the different production methods of each flour could affect the results,
increasing the possible error. To solve this problem, it would be necessary to make sure that the flours used come
from the same brand that provides a variety of options. This way, the production methods will be the same, or at
least very similar, making the reaction more controlled as all types of flour have similar components. Finally,
because of the level of this investigation, access to sources was limited to completely sustain the results of almond
flour producing big amounts of 𝐶𝑂2 which led me to obtain little explanations about it. Hence, It would be
necessary to have access to further academic resources with specialized information regarding different types of
nuts and fruits that can provide more informed and sustained information about the components of almonds. This
would also be beneficial for the rest of the flours since it could provide better explanations for the results of the
investigation. If the experiment is conducted at a professional level, the availability of sources would be greater and
the investigation would be better sustained than what is possible now.
4.4 Recommendations for Future Investigations
For further investigations, it is recommended to increase the number of flours, in order to review the different
effects on yeast fermentation in regard to all its different components. It would provide a greater understanding of
further grains, seeds, and fruits and their positive or negative effect on cellular respiration rates. Additionally, it
would also provide a broader perspective on the topic if more variables were considered apart from the dough’s
height variation and production of carbon dioxide. This would allow more information about each flour on other
effects produced by the different components and the way they react with yeast, for example, on the weight of the
dough. Finally, it would be beneficial to seek deeper explanations for the effect of each flour that does not directly
relate to the glycemic index or the gluten present. Conducting further research on the matter would allow a more
profound analysis and, therefore, understanding of the broader elements inside each flour and also the components
of the yeast itself and its effects, finding other explanations that are not already shown in my investigation.
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ormation%20and%20properties

6. APPENDIX

Appendix. 1 Appendix. 2 Appendix. 3 Appendix. 4

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