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Introduction
The fresh and saltwater ecosystems of Earth are incredibly delicate
systems that require precise ecological balance to thrive. One of the most
important contributors to this balance is algae. Algae are diverse organisms that
primarily obtain energy through photosynthesis and carbon absorption. They play
an important role in the carbon cycle that takes place in oceans and lakes: during
photosynthesis, algae absorb dissolved carbon dioxide in the water; when the
algae is eaten or eventually dies, it releases its store of carbon back into the
ecosystem. Algae are so important because they regulate the carbon dioxide
levels in their ecosystems, which directly affects all the other organisms present.
Unfortunately, global warming is having a negative effect on the algae.
Due to global warming, caused by an increase in the amount of greenhouse
gases in the atmosphere, oceans and lakes are becoming more and more acidic.
Since the Industrial Revolution, these bodies of water have become 30% more
acidic (“Ocean Acidification”). This dramatic change in pH may affect the algae in
some previously unknown way. Determining if there is an effect – and if there is,
how significant the effect is – is crucial to protecting algae from changes in its
environment. If acidic ecosystems cause algae to die faster and in larger
quantities, then greater amounts of carbon dioxide will be released into the water
than ever before, potentially putting entire ecosystems at risk.
In order to determine the effect of pH on the amount of carbon dioxide
absorption, samples of algae were placed in environments of different pH levels.
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The carbon dioxide levels inside the environments were measured, and after two
days they were measured again to determine the change in concentration. By
analyzing the change in carbon dioxide concentration over two days, which
environment causes the algae to absorb more carbon dioxide was determined.
By establishing what the problem will be now, solutions can be created
now that will work in the future. If the problem of ocean acidification and
increased carbon dioxide levels in the ocean can be solved soon, it will be less of
a problem later. With a solution already in hand, scientists can be confident that
when global warming finally brings the pH of water ecosystems down to an
extremely dangerous level, they will already have a way to counter its effects and
protect the ecosystems from further damage.
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Review of Literature
The world is currently plagued by an increase in carbon dioxide production
from man-made sources. In the air, it captures heat entering the atmosphere; in
the oceans, it disrupts the carbon cycle and is detrimental to the growth and
development of aquatic life. Almost half of the gaseous carbon dioxide produced
by humans is absorbed by the lakes, oceans, rivers, and any other permanent
bodies of water (Fabry). This process is helpful to humans because it slows the
climate change that would occur if the carbon dioxide stayed in the air, but once
dissolved in the ocean or lake, the carbon dioxide goes through a process called
the carbon cycle. Since the industrial revolution and the advent of global
warming, this cycle has changed so that it results in more free hydrogen ions,
increasing the acidity of the water (see Figure 1). Being the large bodies of water
they are, oceans have absorbed a vast amount of carbon dioxide and increased
their acidity from 8.2 on the pH scale to 8.1 (Doney et al). Acidity is measured on
the pH scale, or the concentration of hydrogen cations in a solution. The pH
scale is a logarithmic scale and ranges from 0 to 14, 0 being the most acidic and
14 being the most alkaline. Because it is logarithmic, each value gone up or
down along the scale is a ten-fold increase in either acidity or alkalinity. The
increase in pH may not seem like much, but the pH scale is logarithmic, meaning
that just one-tenth of a point means a 40% increase in acidity.
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Figure 1. Chemical Process of Ocean Acidification
Figure 1 shows how carbon dioxide and free carbonate ions combine with
the water they are in to form bicarbonate ions, which then split into H +ions,
causing the pH to increase. Algae plays the role of absorbing the carbon dioxide;
the problem arises when the algae dies and the carbon dioxide is released into
the cycle. The increase in free carbon dioxide when the algae dies means even
more hydrogen ions are produced at the end of the cycle.
This experiment was designed to address the algae in freshwater
ecosystems. In these freshwater environments, acidification still occurs like it
would in oceans, just at a slower rate than in oceans because of the drastic
decrease in size; about 0.0075% of the total water on the earth is fresh and on
the surface, able to interact with atmospheric carbon dioxide (Perlman). The
effect of acidification is even greater in freshwater ecosystems because they sit
they both rest on and pass over sediment, which is generally already acidic.
Luckily, there is one type of organism that photosynthesizes the carbon dioxide
entering the lakes: algae. Through the process of photosynthesis, algae can
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absorb some of the aqueous carbon dioxide present in the water to turn into free
oxygen that is then released into the environment.
The experiment that was conducted was one that tests how well algae can
photosynthesize carbon dioxide in water when subjected to more acidic and
alkaline environments. During photosynthesis, plants intake carbon dioxide,
water, and energy from the sun and create sugars and oxygen (Figure 2). The
experimental design at hand is based on one conducted by Mr. Azov, a
researcher at Israel Oceanographic and Limnological Research, which
determined how much carbon dioxide was taken up from the surroundings by
algae when grown in high and low carbon dioxide rich environments by growing
them in either acidic or alkaline environments and then switching their
environments to the other pH. This experiment is very similar to the one that was
conducted. The main difference was that in the experiment at hand, there was no
difference in intial carbon dioxide concentration and the algae stays in one pH for
the whole duration of the experiment.
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Figure 2. Photosynthesis Chemical Equation
Figure 2 shows the chemical equation for photosynthesis that occurs in
chlorophyll of plants.
Algae is a plant without stems or leaves, but still contains chlorophyll and
feeds itself by producing its own sugars for energy. Algae can be found in both
marine and freshwater environments. Algae requires high amounts of
phosphorus, P and nitrogen, N2 to begin growing (“Information About Why Algae
Grow In Lakes”). Algae is very important in its ecosystem because it makes up
the base of the local food chains – it is eaten by the herbivores – and it is the
primary producer – it makes its own food – in its ecosystem. More importantly, at
least on a basis of the experiment that was conducted, algae is a plant that can
photosynthesize carbon dioxide, sunlight, and other nutrients into water, oxygen,
and sugar for energy in the plant, as shown in Figure 2. It was used in this
experiment because of how much its environment is affected by the acidity and
alkalinity. Photosynthesis occurs in two different reactions: light and dark. This
“light” reaction requires sunlight to create ATP and other nutrients required to run
the “dark” reaction. The “dark” reaction does not require sunlight to complete,
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and it converts carbon dioxide to glucose and free oxygen by stripping the carbon
dioxide of its carbon to form glucose and leaving oxygen as a byproduct (Carter).
Photosynthesis is aided by the protein ribulose bisphosphate
carboxylase/oxygenase (RuBisCo), which “[creates] organic carbon from the
inorganic carbon dioxide in the air” (Goodsell). This process is advantageous to
humans because over time it can reduce the amount of carbon dioxide in the
water and air which will help control the climate and allow for the water to
become less acidic. Algae is a very robust plant, so it should be able to handle
the changes to its environment very well. This robustness was a large factor in
why algae was chosen to be tested on. Since its environment is changing so
drastically, its ability to survive was tested.
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Problem Statement
Problem:
Does the pH of Spirogyra’s environment affect how much CO2 it can
absorb and photosynthesize?
Hypothesis:
A higher pH, which means a more alkaline solution, will result in more CO2
consumption by the Spirogyra culture.
Data Measured:
The independent variable was the pH of the solution Spirogyra was in
during trials, ranging from acidic at 5.5 to alkaline at 8.5 on the logarithmic pH
scale. The dependent variable was the CO2 concentration of the environment
after Spirogyra was introduced and was measured in ppm (parts per million). The
data was measured with a Vernier CO2 gas sensor. The Oxygen (O2)
concentration was measured in parts per trillion (ppm) using a Vernier O 2 Gas
Sensor. The data was interpreted using a two-sample t-test because two
independent samples (the acidic and the basic samples) were compared.
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Experimental Design
Materials:
0.1 M hydrochloric acid, HCl
0.025 M sodium borate, Na2B4O7
0.1 M trisodium citrate, Na3C6H5O7
0.1 M citric acid, C6H8O7
(1) 250 mL Erlenmeyer Flask
Vernier CO2 Gas Sensor 0 – 10,000 ppm
Vernier O2 Gas Sensor 0 – 270 ppt
(10) 250 mL Nalgene sampling bottle
(3) 10 mL Graduated Cylinder
Spirogyra algal culture
Deionized water
(2) 500 mL beaker
Vernier pH Sensor
Vernier LabQuest
Procedure:
1.
Prepare citric acid – trisodium citrate buffer solution for tests involving
acidic environment (see appendix A).
2.
Prepare hyrdrochloric acid – sodium borate buffer solution for tests
involving alkaline environment (see appendix A).
3.
Prepare Vernier LabQuest with CO2 and O2 gas sensor attached (see
appendix).
4.
To randomize, use the random integer function on the TI-nSpire calculator
and assign each group a number (“1” for acidic tests, “2” for neutral tests,
“3” for alkaline tests, “4” for control tests). Since the algae is coming from
a single population, it is already random to pull out samples.
Note: The acidic, alkaline, and neutral tests should use three bottles
each, while the control only uses one.
5.
Prepare the ten gas sampling bottles
Place the bottle on its side. Aliquot 20 mL of the appropriate water
to cover the new bottom (see Figure 1.). Record the CO2 and O2
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concentrations before introducing the algae. Introduce the algal
culture into the water (if applicable). Record the CO2 and O2
concentrations. Close the bottle with the lid.
6.
Record the CO2 and O2 concentrations at the beginning and end of the 48
hours.
7.
Clean out bottles and repeat steps 3-6 two more times for a total of 9 trials
for each treatment and 3 control trials.
Diagram:
Figure 3. Diagram of Bottle Setup
Figure 3 is a diagram of the bottle setup. The Nalgene bottle was filled
with 20mL. The algae sample was then placed inside the bottle and the cap was
closed tightly.
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Data and Observations
Table 1
Carbon Dioxide Concentration Trials Data
Set
Trial
pH
1
1
2
3
4
5
6
7
8
9
10
5.5
5.5
5.5
7.0
7.0
7.0
8.5
8.5
8.5
405
700
523
383
688
473
118
84
96
5410
5010
4605
601
875
717
110
50
50
5380
4088
4315
850
765
760
53
48
49
Change in CO2
Concentration
(ppm)
4975
3388
3792
467
77
287
-65
-36
-47
7NA
553
756
654
101
5.5
5.5
5.5
7.0
7.0
7.0
8.5
8.5
8.5
505
400
428
390
580
530
206
210
206
649
534
386
367
576
583
222
242
256
351
356
352
410
378
530
220
214
170
-154
-44
-76
20
-202
0
14
4
-36
7NA
420
408
418
-2
5.5
5.5
5.5
7.0
7.0
7.0
8.5
8.5
8.5
205
190
700
190
248
205
160
260
169
533
775
1490
373
180
155
47
47
48
1550
1512
1045
541
445
363
136
144
143
1345
1322
345
351
197
158
-24
-116
-26
7NA
230
200
138
-92
2
3
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
CO2
Concentration
Before (ppm)
CO2
Concentration
During (ppm)
CO2
Concentration
After (ppm)
Table 1 lists the raw data of the Carbon Dioxide Trials. Every 10 trials are
separated into a set; in each set, 3 trials are acidic (pH of 5.5), 3 are neutral (pH
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of 7), 3 are basic (pH of 8.5), and 1 is a control of just water (pH of 7) (7NA refers
to No Algae). The Carbon Dioxide concentration is measured in parts per million
(ppm). The change in concentration is simply the final CO2 level minus the initial
CO2 level.
The acidic trials in sets 1 and 3 are abnormally high. Because the other
trials in those sets are similar to the rest, it is safe to assume that whatever
happened in the acidic trials had no effect on the other trials.
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Table 2
Oxygen Concentration Trials Data
Set
Trial
pH
O2 Concentration
Before (ppt)
O2 Concentration
During (ppt)
O2 Concentration
After (ppt)
1
1
2
3
4
5
6
7
8
9
10
5.5
5.5
5.5
7.0
7.0
7.0
8.5
8.5
8.5
177.12
176.7
177.08
176.82
176.49
177.18
176.9
177.65
175.85
149.2
149.5
148.7
148.29
148.3
148.7
148.8
148.05
147.95
145.26
146.23
145.99
146.73
146.7
146.9
146.98
146.83
146.74
Change in O2
Concentration
(ppt)
-31.86
-30.47
-31.09
-30.09
-29.79
-30.28
-29.92
-30.82
-29.11
7NA
176.89
148.5
146.52
-30.37
2
11
12
13
14
15
16
17
18
19
20
5.5
5.5
5.5
7.0
7.0
7.0
8.5
8.5
8.5
14.25
14.28
14.33
14.37
14.45
14.55
14.44
14.54
14.6
15.26
15.15
15.09
14.93
14.85
14.9
14.85
14.68
14.68
14.6
14.6
14.35
14.33
14.31
14.33
14.28
14.28
14.41
0.35
0.32
0.02
-0.04
-0.14
-0.22
-0.16
-0.26
-0.19
7NA
14.55
14.6
14.36
-0.19
3
21
22
23
24
25
26
27
28
29
30
5.5
5.5
5.5
7.0
7.0
7.0
8.5
8.5
8.5
14.42
14.34
14.27
14.27
14.44
14.43
14.41
14.43
14.36
14.17
14.23
14.13
14.42
14.45
14.43
14.26
14.34
14.42
12.51
12.51
12.57
12.55
12.56
12.47
12.6
12.69
12.58
-1.91
-1.83
-1.7
-1.72
-1.88
-1.96
-1.81
-1.74
-1.78
7NA
14.23
14.42
12.61
-1.62
Table 2 lists the raw data of the Oxygen concentration trials. Oxygen
concentration was measured in parts per trillion (ppt). The change in
concentration is the final concentration minus the initial concentration.
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The trials in the first set had much larger changes in concentration than in
sets 2 and 3. The trials in each set are consistent with each other, though, so any
error in the sets also must have stayed consistent.
Table 3
Table of Obersvations
Date
Observations
29-Oct
30-Oct
31-Oct
1-Nov
11-Nov
12-Nov
13-Nov
14-Nov
15-Nov
17-Nov
Made more Acid Buffer Solution, measured O2 and CO2 concentrations before; Room is very
hot, Acidic solution is very cloudy
Room is hot
Slight cloudiness in acidic bottles; Condensation inside all bottles; algae not all the way in
bottle 8.5-3;
Acidic algae may have died, releasing all of their CO2
Acidic algae is practically white, definitely dead; Nothing else very important
Filled up bottles with all buffer solutions, no algae yet
Measured before O2 and CO2 levels for set 2; putting algae in bottles, 5.5 - 3 lost a small
amount of water when opening bottle; Unsure if using same O2 as before
Seperated algae using Buchner funnel; found small insects living in water;
No observations/nothing notable
Finished 2nd set, began 3rd; Using a buchner funnel again; same O2 as Set 2
pH 7 - 3 bottle's mouth is slightly larger than the others, possible leak when measuring
Every O2 trial bounced within a range instead of staying steady like normal; taken a day later
than the rest
Acidic trials slightly cloudy again
Table 3 lists the observations made during data collection. Any significant
or unexpected events and factors were logged each day. The bold lines separate
each set of trials (e.g. November 11-13 was Set 2).
During the first set of trials, the room was hotter than normal due to
another experiment going on in the room; this may have affected the algae, and
therefore the data, in some way.
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In the second and third sets, a Buchner funnel was used to grant easier
access to the algae; this method was not used in set 1 and although there are no
obvious effects it may have had, the funnel method may have had some unseen
effect on the algae. Additionally, a different O2 sensor was used in Set 1 than in
Sets 2 and 3; after the first set, the sensors were packed up and put away. At the
beginning of Set 2, which sensor was used before could not be determined. The
same sensor was used in Sets 2 and 3.
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Data Analysis and Interpretation
For this experiment, algae’s ability to absorb CO2 when subjected to
different pH levels was measured. To find the amount absorbed, algae was
placed in a closed system and allowed to live while subjected to different pH
environments. The CO2 and O2 concentrations were measured using Vernier
CO2 and O2 gas sensors. The concentration of O2 was measured in parts per
trillion (ppt), while the concentration of CO2 was measured in parts per million
(ppm). These concentrations were measured because CO2 is absorbed by algae
during photosynthesis and O2 is released as a byproduct. An increase in O2
and/or a decline in CO2 are tell-tale signs that the algae are living and are
continuing the photosynthetic processes. This data was analyzed with a two
sample t-test, comparing the acidic/alkaline environment’s changes in
concentrations to the neutral environment’s changes in concentration. Any
significant differences (positive for O2 and negative for CO2) would signify that
the algae are living. If the differences are significantly in the opposite direction, it
could be concluded that the algae are not living or that some other lurking
variable skewed the data severely. The data that was collected is reliable
because of randomization. This randomization reduces bias that may have been
present. The data is also reliable because controls were run; in every set of 10
trials, one bottle contained no algae and a neutral environment. Because these
controls had no algae in them, they underwent the same treatment and
experienced the same lurking variables as the other trials. Therefore, because
the controls showed little variability, it was safe to assume that the data collected
was reliable.
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The data that was collected was interpreted using a two sample t-test (see
Appendix B). This t-test compared the alkaline and acidic environment trials to
the neutral environment trials. First, two hypotheses were created:
𝐻0 : µ = 0
𝐻𝑎 : µ > 0
The first, called the null hypothesis (H0), determines whether the treatment had
no effect; the second, called the alternative hypothesis (Ha), determines if the
treatment did have an effect. So if at the end of the test the null hypothesis is
rejected, it can be concluded that pH did have a significant effect on the CO 2
absorption of algae. In doing this comparison, p-values were calculated. These
calculated p-values were then compared to the alpha level of 0.05. If the alpha
level was exceeded, then the treatment’s effect could be considered insignificant
because the effect could be accounted to simple chance alone. However, if the
alpha level was not exceeded, then the treatment’s effect would be considered
significant and be further explored.
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Table 4
The p-values for Every t-test Performed
t-test
Trial
Run
Concentration
CO2
1
O2
CO2
2
O2
CO2
3
O2
Treatment
5.5
8.5
5.5
8.5
5.5
8.5
5.5
8.5
5.5
8.5
5.5
8.5
P-Value
0.001520083
0.044669167
0.063484199
0.8504915
0.714479162
0.492953335
0.036515616
0.307506195
0.083309298
0.011819341
0.690488853
0.355269261
Significance
yes
yes
no
no
no
no
yes
no
no
yes
no
no
Table 4 shows the p-values of every set of trials and if they were
significant. In accordance with Table 4, there were only 4 significant effects: the
effects of both treatments on CO2 concentration in the first trial run, the effect of
acidity on O2 concentration in the second trial run, and the effect of alkalinity on
CO2 concentration in the third trial run. All of the significant effects agree with
current knowledge in the field, with the exception of the effect of acidity on CO 2
concentration in the first trial. This means that the CO2 decreased significantly
and O2 increased significantly when the algae was exposed to an acidic
environment. This knowledge can be applied to the original hypothesis of a more
alkaline solution resulting in a better environment to harbor algae. Because of
consistently small p-values for the alkaline environment’s effect, H0 was rejected.
This means that high pH had a statistically significant effect on algae’s ability to
absorb CO2.
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Graph of O2 Standards
Concentration (ppt)
0
-5
1
2
3
4
5
6
7
8
9
-10
-15
Change in O
-20
-25
-30
-35
Trial
Figure 4. Graph of O2 Standards
Concentration (ppm)
Graph of CO2 Standards
600
500
400
300
200
100
0
-100
-200
-300
Change in CO
1 2 3 4 5 6 7 8 9
Trial
Figure 5. Graph of CO2 Standards
The graphs in figures 4 and 5 show little variance in the data, so it was
safe to assume that the data collected was reliable.
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Figure 6. CO2 Trials Box Plots
The high and neutral plots are relatively far apart, meaning that there was
most likely a relationship between the two. The low plot’s median is far away
from neutral, but this is most likely due to extreme data points in the data.
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Figure 7. O2 Trials Box Plots
The plots have a lot of overlap and there medians are all very close
together, meaning that there was very little chance that oxygen levels were
affected by the changes in pH.
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Conclusion
The purpose of the research was to determine if the acidity of algae’s
environment has a significant effect on how well algae absorbs carbon dioxide
(CO2). To conduct this experiment, algae was placed in bottles partially filled with
buffer solutions of either a basic (8.5) pH, neutral (7) pH, or acidic (5.5) pH and
allowed to photosynthesize and absorb CO2. These levels were then measured
with Vernier CO2 sensors and the data was analyzed with multiple two sample ttests. The change in the amount of CO2 gas was measured in the different acidic
and alkaline environments and compared to the change in CO2 gas levels in a
neutral environment. Consistently, the alkaline environment significantly
decreased the CO2 levels and increased the O2 levels.
All things considered, the original hypothesis of an alkaline environment
would cause the algae to absorb more CO2 was accepted. In two of the three trial
runs, the CO2 levels were decreased significantly in the alkaline environments.
According to the experiment conducted by Mr. Azov, “pH values in the range of 8
to 9 were important [when growing algae]”. This means that algae grows better in
high pH environments. Since it grows better, it makes sense that the CO 2 would
increase because the photosynthesis process requires algae to absorb CO 2,
water, and sunlight to create O2 and sugars for itself. Even if the O2
concentrations did not increase as significantly as the CO2, the most important
factor was the reduction in CO2 concentration, because high CO2 concentrations
can cause major problems in the ecosystem. High CO2 levels have been linked
to problems with the neurological transmitters in fish, causing their populations to
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drop (Nilsson et. al.). This information could be used as a basis for further
experimentation on this topic, or it could account for a possible lurking variable in
other experimentation.
A few abnormalities were encountered during the experimentation
process. The most important one being the extreme increases in CO2 for the
acidic trial runs. These results can be attributed to the fact that the algae died.
When algae dies, it releases all of its photosynthetic reactants it currently has.
This was to be expected.
All of these results can be applied to the world. With the acidic trials
(where the concentration suddenly increased), biologists can be sure to watch
the environment so as to keep the algae from dying and releasing its CO2 into its
environment, which could be dangerous for the other organisms living with it. In
the other direction, the knowledge that algae absorbs more CO 2 in a basic
environment could help scientists conducting labs on algae keep the algae alive
longer and possibly better. This is supported by the fact that a carbon dioxide
fixing enzyme RuBisCo – which helps the algae absorb and utilize the carbon
dioxide – works best in a pH of 8 (Buehler). The data collected and conclusions
drawn support other research done in this field; namely, an experiment
performed by Schrock, Vallar, and Weaver, students at the University of
Missouri, concluded that a more acidic environment is detrimental to the
photosynthetic process of aquatic plants. For future research, the effects of other
buffer solutions could be tested to ensure that the chemicals in the buffer
solutions did not play a major role in helping or inhibiting absorption.
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Appendix A
Preparing Buffer Solutions:
To determine how many grams needed (citric acid):
grams=molecular weight×volume×molarity
grams=192.124 gmol×0.02325 L×0.1 M
grams≈0.446
To determine how many grams needed (trisodium citrate):
grams=molecular weight×volume×molarity
grams=258.06 gmol×0.07675 L×0.1 M
grams≈1.980
Combine the 23.25 mL of citric acid with the 76.75 mL of trisodium citrate to
create 100 mL of solution. Measure the pH to ensure it is 5.5
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Appendix B
Two-Sample t-Test
1
𝑡=
−
2
𝑠1
𝑠2 2
+
𝑛1
𝑛2
√
2
Above is the equation used to calculate the t-value in a two-sample t-test.
is
the sample mean, s is the sample standard deviation, and n is the sample size.
The subscripts indicate which population the value comes from (e.g. s1 is the
sample standard deviation from population 1, s2 from population 2). Once a tvalue is calculated, a p-value must be found using either a calculator or a p-value
table.
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Works Cited
Azov, Y. "Effect of PH on Inorganic Carbon Uptake in Algal Cultures." Applied
and Environmental Microbiology 43.6 (1982): 1300-306. American Society
for Microbiology, June 1982. Web. 2 Oct. 2013.
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