The purpose of the experiment was to determine which invasive seaweed, brown Sargassum or green Ulva Lactuca, could produce more bioethanol fuel over time. It was hypothesized that Sargassum would produce the most fuel after 144 hours of fermentation. Both seaweeds were processed and fermented for 96, 120, and 144 hours. Absorbance values indicated more glucose and ethanol were produced from Sargassum over time, supporting the hypothesis. While an estimation, more biofuel was calculated from Sargassum, showing potential practical use in reducing pollution from seaweed blooms. Limitations included ethanol amounts being estimates and seaweed composition varying by collection time.

1. The Effectiveness of Invasive
Types of Seaweed Species, Brown
(Sargassum) and Green (Ulva
Lactuca) as Bioethanol fuels
By: Maya Volot

2. Abstract
The purpose of this experiment is to test which invasive type of seaweed species, Brown (Sargassum) or
Green (Ulva Lactuca) can produce the most Bioethanol fuel at a given time of fermentation. If a Sargassum
biofuel and an Ulva Lactuca biofuel are created then the Sargassum biofuel will be estimated to produce the
most biofuel after 144 hours of fermentation. To perform this experiment, Both seaweeds were cleaned
with distilled water. They were then dehydrated and set aside. After turned into fine powders the seaweeds
were hydrolyzed with sulfuric acid and autoclaved. Cellulase and Amylase were added and enzymatic
digestion was performed in an incubator. Yeast was then placed in the solutions to ferment and ethanol
yield would be calculated (96h, 120h, 144h). Samples were then diluted using distilled water and Sulfuric
acid. Samples of each solution were then placed into a microplate and placed into the microplate reader to
measure absorbance value. With that number and a series of calculations the amount of glucose was
calculated and then the yield of bioethanol produced was estimated. The amount of ethanol calculated in
grams for the green seaweed was 0.001096937, 0.001225892, and 0.001382597. While, the amount of
ethanol calculated in grams for the brown seaweed was 0.001222627, 0.001425038, and 0.001607861. The
longer the seaweed mixtures fermented the more ethanol was yielded. This experiment is of practical use
because this biofuel can reduce pollution as well as alleviate the coastal issue of these invasive species and
many more others in the future.

3. Purpose:
The purpose of this experiment is to test which invasive type of
seaweed species, Brown (Sargassum) or Green (Ulva Lactuca) can
produce the most Bioethanol fuel at a given time of fermentation.
Hypothesis:
If a Sargassum biofuel and an Ulva Lactuca biofuel are created
then the Sargassum biofuel will be estimated to produce the most
biofuel after 144 hours of fermentation.

4. Background Research
Seaweed Background:
● Sargassum, is commonly seen on coastal areas and beaches, it is a genus of large brown algae grown in both tropical and
temperate oceans.
● It is a type of seaweed that floats and can become a large surface mat floating on the ocean surface (Gramling and
Carolyn, 2019). These mats provide shelter, protection from predators, and allow camouflage to marine species(Coston-
Clements et.al, 1991).
● It is one of the most common species of invasive seaweeds has become a serious issue in the United States Gulf, Atlantic
Ocean etc. shorelines throughout spring and summer months. These mats suffocate corals and sea grasses (Cotton-
Clements et.al, 1991). Left on coastlines it can release toxic gases to humans such as ammonia and hydrogen sulphide,
potentially causing health problems such as upper airway irritation, headaches, and vestibular syndrome (“Invasive
Species; Findings In”, 2018; Resiere et.al, 2018).
● Ulva Lactuca also known as the “sea lettuce” is a species of macroalgae that grows relatively everywhere, it is sheet like,
light green and translucent.
● The seaweed mostly grows on rocks as well as float on the surface of the ocean. It is also an important food source for
grazing marine life such as crustaceans that can not get their food from the surface of the ocean (“What is Sea
Lettuce?”, n.d.).
● Beaches of northern France were being invaded by a green tide of potentially toxic sea lettuce that killed two men in
under a week in the Morlaix Bay and the Douarnenez Bay in Brittany. They died because of a possible hydrogen
sulphide poisoning because of the decomposure of Ulva Lactuca just like sargassum does to people.

5. Seaweed as a Biofuel
● Seaweeds are a great way to decrease the amount of pollution while still being able to use
the fuels we need for our society to function. they contain 85-90% of water which is very
suitable for processes like anaerobic digestion to make biogas and fermentation to make
ethanol.
● They contain high amounts of carbohydrates and low amounts of lignin because seaweed is
an aquatic biomass, terrestrial biomasses usually contain high amounts of lignin compared
to aquatic ones which is perfect for making bioethanol as it eases the process of breaking
down the seaweed (Pros and Cons Seaweed, n.d.).
● Not only do Fossil fuels take millions of years to replenish, they also release an abundant
amount of CO2 into the atmosphere which is a greenhouse gas that leads to global warming.
Burning these organic materials do not increase the carbon emissions produced because
they consume CO2 when growing so they are simply putting back out what they took in
(Luterbacher, C. and Luterbacher, J., 2015).

6. Processes of Extracting Biofuels
Acid/Enzymatic Hydrolysis:
● Acid hydrolysis is a process in which
dilute acid at high a temperature and
pressure is used. Another type of
hydrolysis called enzymatic hydrolysis
is where cellulose chains are broken
into glucose molecules by cellulase
and/or amylase enzymes. With both
processes combined, it creates a
faster and more efficient way to
produce bioethanol from seaweeds
(Basu, 2018).
Fermentation:
● In biochemistry, Fermentation is known
as the process of extraction of energy
from carbohydrates in the absence of
oxygen. This process is usually helped
by yeast. The yeast helps with the
process by breaking down the sugars in
the seaweed and releasing ethanol and
CO2 as waste products. Cellulase and
Amylase are also aiding enzymes in this
process that help break down the
cellulose in the cell walls of green plants
( Shobharani et.al, 2012).
Microplate Reader:
● Gas chromatography is a
common type of
chromatography used in
analytical chemistry for
separating and analyzing
compounds that can be
vaporized without
decomposition.
Math Equations:
● are instruments which are used to detect
biological, chemical or physical events of
samples in microtiter plates. Absorbance is a
measure of the quantity of light absorbed by a
sample which was measured in this experiment.
Gas Chromatography:
Key:
C = concentration in units of moles per liter
Epsilon = molar absorptivity
l = length
e mol = moles of ethanol
v = moles of glucose
A = absorbance

7. Methods
● (4) Disposable gloves
● (1) Safety goggles
● (1) Precision scale
● (3) Weigh boat
● (100g) Sargassum
● (1) Sink
● Distilled water
● (1) Paper towel roll
● (4) Underpads
● (1) dehydrator
● (1) Food processor
● (2) glass bottles
● (1) scoopula
● (3) sticker label
● (1) permanent marker
● (1) Soap bottle
● (100 g) Ulva Lactuca
● (2) Saccharomyces cerevisiae cultures
Materials:
● (40g) Yeast Malt Agar
● (1) glass stirring rod
● (1) autoclave
● (1) incubator
● (100 mL)Bleach
● (4) graduated cylinder
● (6) graduated pipettes
● (100mL) sulfuric acid 1M
● (50mL) sulfuric acid 18M
● (80mL) Sodium Hydroxide
● (10) pH testing strips
● (10g) Cellulase Enzyme
● (10g) Amylase Enzyme
● (1) Fume Hood
● (1) Hot Plate
● (3) 100 mL beaker
● (6) 150 ml volumetric flask
● (1) Microplate
● (1) Heat resistant gloves
● (1) Microplate Reader

8. Procedure:
1. Wear disposable gloves and eye protection, layout an underpad on the work surface.
2. Wash the measured sargassum thoroughly for about two minutes in a sink with distilled water
3. Place the cleaned sargassum on the dehydrator trays laying them out flat.
4. Place the trays with sargassum into the dehydrator at the lowest temperature setting for about 3-4 hours.
5. After removing the sargassum from the dehydrator, let sit on a safe surface.
6. Place the dried sargassum into the food processor and blend until it becomes a fine powder (about 3-6 min).
7. Using a strainer, sift the powder thoroughly making sure there are no large pieces left into a storage container.
8. Label the container with a permanent marker to write the name sargassum.
9. Clean all the materials used in a sink thoroughly using soap and water.
10. Repeat steps 1-9 with the Ulva Lactuca.
11. Seaweed powder (10% w/v) was then hydrolyzed with 0.2M H2SO4 by autoclaving the solution at 121°C for 20
minutes.
12. The pH was then adjusted to 5.5 using 10M NaOH and a pH testing strip.
13. The cellulase and amylase enzymes (2.5g of each) were then combined to create an enzyme cocktail and added to
the solution.
14. Enzymatic digestion was then performed by incubating the acid-hydrolysed seaweed samples at 50°C with an
agitation at 100 rpm for 18 hours.
15. A malt extract (20 g/l) broth was inoculated with the saccharomyces cerevisiae and incubated at 30 C with shaking at
100 rpm for 18 h to produce seed cultures for fermentation.
16. Fermentation was conducted by fermenting samples in 150 ml Pyrex bottles at 30 C with shaking at 100 rpm.
17. Samples of each seaweed (2g) were collected after 96, 120, and 144 hours of fermentation.

9. Procedure Continued:
1. Transfer each sample after the given amount of fermentation time into two 200 mL beakers & add nearly 100 mL
distilled water to each.
2. Boil the samples between 350-390 C or until boiling separately for 10-15 minutes while stirring on a hot plate.
3. While boiling prepare the phenol red reagent by adding 5g of phenol red into a beaker and 100 mL of distilled water, mix
with a glass stirring rod and place aside until further use.
4. Add 3 mL of conc. H2SO4 into the samples and again boil for 3 mins.
5. Filter the samples into two 250 mL volumetric flask using fluted filter paper
6. Take 1.25 mL from the solutions created above into two 100 mL volumetric flasks and add 100 mL of distilled water.
7. take another 2.5 mL from the above sample into 50 mL conical flask and add 6 mL of conc. H2SO4 and 2.5 mL 5% phenol.
8. Measure the absorbance with respect to the color development at 505 nm using UV-Visible spectrophotometer.
Calculations
1. Using the Beer-Lambert Law calculate the concentration or molarity of each sample (molar absorptivity and path length
are known, and absorbance is the experimental value).
2. Calculate the estimated amount of glucose in moles by multiplying the concentration by the volume of the sample.
3. Using the molar ratios from the fermentation chemical equation, the moles of ethanol are able to be determined (the
moles of ethanol is equal to two times the moles of glucose).
4. By multiplying the amount of moles of ethanol and glucose by their molar masses, an estimated amount of glucose and
ethanol (in grams) will be determined.

10. Results
Data Tables:
Table 1. The absorbance value of each sample.
Table 2. The amount of glucose produced per sample.
Table 3. The amount of ethanol produced per sample.

11. Graphs:
Figure 1. The Absorbance value of each sample.
VENUS
MARS
Figure 2. The total amount of estimated biofuel per sample.

12. Pictures:
90%
70%
50%
70%
40%
90%
Figure 3. The Ulva Lactuca being Dehydrated (Oct.6, 2020). Figure 4. The pH being balanced (Nov.12, 2020). Figure 5. Seaweed Mixtures being Incubated (Nov.12, 2020).
Figure 6. Diluted seaweed solutions being boiled (Nov.19, 2020). Figure 7. Diluted Solutions being filtered (Nov.19, 2020).
Figure 8. Microplate samples (Nov. 19, 2020).

13. Discussion
Conclusion:
The purpose of this experiment is to test which invasive type of seaweed species, Brown (Sargassum) or
Green (Ulva Lactuca) can produce the most Bioethanol fuel at a given time of fermentation. This was tested by
creating two biofuels (one for each seaweed) and recording their absorbance values to first estimate the glucose
content and then the ethanol yield. All data is listed from 96 h, 120 h, to 144 hours of fermentation. The
absorbance values of the green seaweed was 0.672, 0.751, and 0.847. The absorbance values of the green
seaweed was 0.749, 0.873, and 0.985. The amount of glucose calculated in moles for the green seaweed was
1.19055-E05, 1.33051E-05, and 1.50059E-05; In grams that is 6.60845E-08, 7.38534E-08, and 8.3294E-08. The
amount of glucose calculated in moles for the brown seaweed was 1.32697R-05, 1.54665E-05, and 1.74508E-05;
In grams that is 7.36567E-08, 8.58509E-08, and 9.68649E-08. The overall amount of ethanol calculated in moles
for the green seaweed was 2.3811E-05, 2.66102E-05, and 3.00118E-05; In grams that is 0.001096937,
0.001225892, and 0.001382597. The overall amount of ethanol calculated in moles for the brown seaweed was
2.65394E-05, 3.09331E-05, and 3.49016E-05; In grams that is 0.001222627, 0.001425038, and 0.001607861.
If a Sargassum biofuel and an Ulva Lactuca biofuel are created then the Sargassum biofuel will be estimated
to produce the most biofuel after 144 hours of fermentation. The hypothesis was supported, the more time the
seaweed mixtures were given to ferment the more ethanol was yielded.

14. Applications:
This experiment is of practical value because it can help alleviate Florida or
any coastal city having issues of invasion by removing these seaweed masses
from coasts when they come. Instead of letting the blooms rot on coastlines and
potentially medically harm people the blooms can be put to practical use as a
biofuel. It would not only decrease the amount of pollution being released into the
atmosphere but with further research it could potentially be of cheaper costs to
create since the seaweed is not expensive to purchase unlike fossil fuels. The
Biofuel would also help alleviate marine habitats that may have been destroyed
and bring back tourists to help the economy that may have fled because the beach
may have not been enjoyable for them anymore. Knowing that the brown seaweed
had a larger ethanol yield and that the longer the fermentation the more ethanol
produced could help when trying to create this fuel in masses.

15. Limitations:
The main limitation to this experiment is that the exact amount of bioethanol
produced from the samples could not be calculated because of the time and cost
of the machine needed (gas chromatograph). Meaning that the math used to find
the amount of biofuel that will be produced is only an estimation And was based
on the absorbance value of the samples. Another limitation is that these
seaweeds were collected in the month of october where their content of
carbohydrates, lipids, and proteins are not as high, so the ethanol content that
could be produced from these seaweeds would be higher in the summer months
when their carbohydrate, lipid, and protein content is higher. Lastly, To determine
the glucose content of the samples distillation could not be used because of the
standard needed for that process so the glucose content was determined based
on absorbance values and larger samples could not have been produced because
of the size of the microplate.

16. Error Analysis:
Some of the errors that could have occured be in this experiment are that
there could be glucose residues left behind on the container during transferring
of solutions or other confounding variables that were beyond the control of the
researcher. The calculations done with the absorbance values to first find the
glucose content which was then used to estimate the amount of bio ethanol in
each sample could have been calculated wrong if numbers were improperly
rounded etc. Error analysis can be done on the amounts of ethanol produced by
comparing the estimated amounts of bioethanol produced to the actual amounts
that would be produced if gas chromatography could be used. By comparing
these values, one would be able to determine the percent yield of each trial. This
value would allow the researcher to establish an error percentage of their
experiment.

17. Future Research:
To extend this project in the future different amounts, times, and
temperatures could be tested to see what conditions are ideal to make the
seaweeds yield more bioethanol. Other species of seaweeds or types like red
ones that are also invasive could also be tested as well to see if they yield more
biofuel. Different enzymes or hydrolysis techniques could also affect the
outcome of the bioethanol, this could be another experiment performed to
compare enzymes or hydrolysis processes. In the end it is a known fact that
creating biofuels can be very time consuming, so research can be done to figure
out if it is possible to create a seaweed biofuel in a faster and in bulk way so
that it could be sold and used in the real world.

18. Thank you!