The Effects of Carbon Dioxide on Sea Lettuce,

Ulva Lactuca

Molly Lada
Freshman Research Project
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Abstract
Biofuels produced from macroalgae can solve the problem of fossil fuels quickly
depleting. Macroalgae uses carbon dioxide along with other nutrients to gain energy during
photosynthesis. Two tanks of sea lettuce, a type of macroalgae, were set up. One received
supplemental carbon dioxide, and the other did not. It was hypothesized that the sea lettuce in the
tank receiving supplemental carbon dioxide would have a faster growth rate and greater
production of chlorophyll than the sea lettuce that did not. After one month, two sets of samples
from both tanks were tested. A handheld chlorophyll analyzer was used to measure the relative
chlorophyll levels of the water. The measures were greater for the carbon dioxide samples in
both sets. A leaf fluorimeter was used to measure the relative chlorophyll levels of the sea
lettuce. The measures were greater in the carbon dioxide samples for both sets as well. The
hypothesis was supported, as the sea lettuce that received supplemental carbon dioxide was
concluded to produce more chlorophyll than the regular sample.
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Introduction
Algae are organisms that are typically found in the water. The microscopic unicellular
algae, called microalgae, are found in marshes or swamps. The very large and multicellular
algae, called macroalgae, are found in ponds or oceans. Algae can be found in almost any aquatic
environment, as well as in soil, snow, or on other organisms. They play a key role in many
ecosystems. Their diverse nature means that they are easy to find and use for commercial
purposes, such as fuel. (Guiry, n.d.)
The main source of energy today are fossil fuels. Enormous amounts of these fuels- oil,
gas and coal- are used each day, and they are unsafe for the environment in many ways. For
example, burning fossil fuels releases many toxins, causing air pollution that can be harmful to
human health. (Union of Concerned Scientists, 2016) They take millions of years to form, so
once we use all of these fuels they are basically gone forever. As the population increases and
new technology spreads, the demand for fossil fuels will only grow. (Ecotricity, n.d.) It is
becoming increasingly possible that humans will deplete these fuels by 2100, so a new form of
energy is necessary.
Oil found in the cells of algae can easily be transformed into biodiesel. First, the oil is
extracted by either pressing it out, using ultrasonic waves, or chemical solvents. Then, it
undergoes transesterification, where substances like alcohol are added to the oil to create a
reaction. A mix of biodiesel and glycerol is formed from this reaction. Finally, the glycerol is
removed, leaving biodiesel that can be used as fuel. (Cassidy, 2010) Algae is a promising
alternative energy source, so growing more in a shorter amount of time is a priority.
Most algae are autotrophs, meaning they grow through photosynthesis. They convert
sunlight, carbon dioxide and other nutrients into energy which they store as oil, and produce
oxygen as a result. (All About Algae, n.d.) Carbon dioxide is necessary to the growth of algae, so
a large supply is needed. This can be created using yeast and sugar. When the two substances are
mixed, the yeast eats the sugar and the byproduct is carbon dioxide. (Hewitson and Hill, n.d.)
The hypothesis is that if supplemental carbon dioxide is added to sea lettuce its chlorophyll
levels will increase, because its rate of photosynthesis will increase.
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Objective
To determine if the addition of supplemental carbon dioxide to sea lettuce will increase its
photosynthesis rate and levels of chlorophyll.

Hypothesis
I predict that the algae receiving supplemental carbon dioxide will grow quicker and
more abundantly than the algae that is not, because increasing carbon dioxide will increase the
rate at which photosynthesis takes place in the algae.

Methodology
Setting Up the Tanks
1. Two 5 gallon tanks filled with 4 gallons of water with added salt for a salinity of 25 ppt
2. Bubbler placed in both tanks with lamps above the tanks
3. Temperature kept between 72 and 80 °F
4. pH kept between 8.0 and 8.5, monitored using pH strips
5. One piece of ​Ulva​ ​lactuca​ added to each tank
6. Carbon dioxide measured using a carbon dioxide kit
7. Parameters measured every week to keep constant levels
Generating Carbon Dioxide and Analyzing
1. Tubing threaded through cleaned 2-liter bottle cap for transferring CO₂
2. 2 cups of sugar and ½ teaspoon of yeast poured into bottle
3. Warm water poured over the sugar and yeast
4. 3 inches of space are left in the top of the bottle before shaking
5. Cap with tubing is put on, starting in the 3 inches of space inside of the bottle, ending
directly in the water of the second algae tank
6. CO₂ is measured with a carbon dioxide kit and salinity, temperature, and pH are kept
constant
7. CO₂ produced for 3 weeks
8. A new bottle is set up after the third week so that the second tank of algae receives
carbon dioxide for exactly one month
9. Sea lettuce in both tanks grows for one month
10. CO₂ tested again after one month
11. Supplemental CO₂ to the second tank is stopped after one full month
12. Water tested with fluorimeter for relative chlorophyll levels
13. Sea lettuce tested with a Hansatech Pocket PEA Chlorophyll Fluorimeter to find its
chlorophyll levels
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Figure 1- The two tanks from above. A thermometer and bubbler are attached to the inside of
each tank. The sea lettuce receiving supplemental carbon dioxide is on the left, and the regular
sea lettuce is on the right.

Figure 2-​ The 2 liter bottle the carbon dioxide was produced in. The tube ran from inside of the
bottle to the water of the second tank where the supplemental carbon dioxide was added.
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Figure 3- The tube, from the bottle that is producing carbon dioxide, in the water of the second
tank. A bubble of carbon dioxide can be seen at the end of the tube where it is being added into
the water.

Figure 4-​ The sea lettuce and water samples in the MATES lab for testing.
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Figures 5 and 6-​ Using the Hansatech Pocket PEA Chlorophyll Fluorimeter to find the
chlorophyll level of the sea lettuce sample.

Results
Of the relative chlorophyll levels of the water from both sample sets, the samples that
received supplemental carbon dioxide had a higher level. The levels of both regular water
samples ranged from .075 to .076. The levels of both carbon dioxide water samples ranged from
.081 to .083. The relative chlorophyll levels of the sample sets of sea lettuce also showed an
increase in chlorophyll production in the carbon dioxide samples. Both regular samples of the
sea lettuce had levels that ranged from .74 to .78. The samples that received supplemental carbon
dioxide had levels ranging from .83 to .88. The data for each sample was averaged to be put into
bar graphs (figures 7 and 8).
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P<0.0001
Figure 7- ​The levels of relative chlorophyll were averaged for each sea lettuce sample.
The bars on the left are the first sample set, and the bars on the right are the second sample set. P
was less than alpha so there is change.

P<0.00001
Figure 8- ​The levels of relative chlorophyll in the water were averaged for each sample.
The bars on the left are the first sample set, and the bars on the right are the second sample set. P
was less than alpha so there is change.
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Discussion
The results concluded that adding supplemental carbon dioxide to sea lettuce increased its
chlorophyll production rate. This is because the sea lettuce could photosynthesise more
efficiently with more carbon dioxide present. It was able to take more in and use it, along with
water and sunlight, to produce more chlorophyll. The regular sample photosynthesized at a
normal rate, so it did not have high chlorophyll levels.
Although there was not a large difference between the levels of the samples, the fact that
each sample set had very similar results was surprising. The chlorophyll levels were very similar
for the two regular pieces of sea lettuce, as well as for the carbon dioxide pieces. The 2 sets of
regular samples had an average of .77 and .75 FV/FM. The carbon dioxide samples had averages
of .87 and .84 FV/FM. The level of chlorophyll in a healthy piece of sea lettuce is .85 FV/FM.
This shows not only that the carbon dioxide samples were healthier, but that the regular samples
were actually unhealthy. This may have affected the results, as if they were completely healthy
the chlorophyll levels would have been higher.
The fact that the chlorophyll production increased when additional carbon dioxide was
added to the sea lettuce can be beneficial to scientists working on alternate fuel sources. The
project can be expanded to see the effects after multiple months, or even the effects if carbon
dioxide is permanently added. If there is a constant increase in chlorophyll the sea lettuce will
grow quicker. This will allow for more sea lettuce to be converted into biofuel over a shorter
amount of time, compared to regular sea lettuce that grows at a normal rate.

Conclusion
The hypothesis was supported by the results after testing the relative chlorophyll levels of
the two sample sets. The sea lettuce that received supplemental carbon dioxide for one month
had a higher chlorophyll level than the sea lettuce that did not. This shows that the amount of
photosynthesis taking place increased.

Acknowledgements
Thank you Dr. Wnek and Mr. Kelsey for your help with my project.
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References
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All About Algae. (n.d.). ​Algae Basics.​ Retrieved 20 Aug. 2017.
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Ecotricity​. ​(n.d.). ​The End of Fossil Fuels.​ Retrieved 20 Aug. 2017
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