STEM is taught by Dr. C. Throughout
the course, we work on our independent research projects until
February Fair, where we have a chance to move on and participate in
statewide and national science competitions. It has been an amazing
experience to develop and run my own experiment, and it has taught me
the importance of time-management and organizational skills. My
project focuses on understanding the environmental effects of an amino
acid, L-lysine, which was shown to kill commonly-appearing harmful
algae cells. I was inspired to take on this topic when the beach I
work at had to close due to a harmful algae bloom. Without all my new
free time, I started researching treatment methods for harmful algae
blooms, only to find out that there really weren't any effective and
eco-friendly methods. Scroll down to learn more about my project!
My Project:
Investigating the effects of L-lysine on M. aeruginosa
and D. pulex
This project examined the effects of amino acid L-lysine on
zooplankton species Daphnia pulex to identify any
ecological consequences of using L-lysine to control harmful blooms
of Microcystis aeruginosa, one of the most common
toxin-producing species of algae. It was found that the lysine
promoted the growth of D. pulex by killing M.
aeruginosa cells, supporting the use of lyisne as a method to
control Microcystis blooms.
Abstract
Harmful algae blooms (HABs) are a rising risk as global temperatures
increase. HABs block sunlight for aquatic life, with some species
producing toxins that harm humans. Current methods to treat HABs,
like physical removal and chemical treatment, are inefficient and
ineffective, having little to no effect on the bloom. Recent studies
have shown that amino acid L-lysine is extremely effective at
killing Microcystis cells, a type of harmful algae. However,
research has not been done to determine the environmental effects
and sustainability of lysine applications. This study examined how
L-lysine would affect the growth of a zooplankton community
undergoing an HAB. It was hypothesized that if L-lysine was added to
a bloom of M. aeruginosa, it would inhibit the growth of
algae while promoting that of zooplankton species D. pulex.
Different concentrations of lysine were added to test groups
containing M. aeruginosa and D. pulex. Over three
days, the test groups were observed to see the effect of lysine on
the zooplankton population. Results were used to determine the
maximum allowable toxicant concentration of lysine, indicating if a
concentration of lysine could be used to manage a bloom without
significantly harming the zooplankton population. Further testing,
such as in situ tests, should be conducted to determine any other
potential effects of L-lysine on the aquatic ecosystem before
real-world applications are implemented.
How will amino acid L-lysine affect the growth of zooplankton with a
bloom of M. aeruginosa?
Hypothesis
If L-lysine is added to a bloom of M.
aeruginosa, it will inhibit the growth of algae while promoting the
growth of zooplankton.
Background
As global temperatures and pollution increase, harmful algae blooms
are a rising threat as these conditions allow algae to grow and form
blooms at unprecedented rates. Algae is a key aspect of the
environment, producing an estimated 70% of all atmospheric oxygen
(“Oxygen levels,” 2022). However, areas affected by climate change
may experience dangerous rates of algal growth, where the algae form
a thick layer across the surface of the water, blocking sunlight and
killing marine life below. When algae grow exponentially like this,
it is called a bloom. Along with blocking sunlight, certain species
of algae can produce toxins that can harm humans as well. These
toxins vary in severity and effects, but they harm marine life,
land-dwelling animals, and humans alike. Beaches all across the
world have lost vast amounts of money as they are forced to close
for weeks at a time when these blooms arise. Small coastal towns are
also devastated as harmful algae blooms pollute their water systems
and shut down the local fisheries. Harmful algae blooms will only
become more frequent as rising temperatures will create even more
favorable conditions for these blooms to arise.
Current
methods used to control harmful algae blooms include physical
removal, chemical algicides, and waiting for the bloom to disappear
on its own (Tian et al., 2018). These methods are highly inefficient
and have even been shown to not have a significant effect on water
quality improvement (Anantapantula et al., 2023). Moreover, chemical
treatments could be detrimental to the surrounding environment if
used without caution or extensive background research. A sustainable
and cost-effective method to control these harmful algae blooms is
of the utmost importance as these blooms pose an increasing threat
on marine life, food supply, freshwater resources, and the economy
as a whole. As most species of harmful cyanobacteria and algae will
require different treatments and procedures, there may not be a “one
size fits all” method that can be applied or solution that can be
added to prevent the growth harmful algae blooms (Errat et al.,
2022). For this reason, some scientists are focusing more on
selective approaches, which will target certain species of algae
rather than looking for a solution which can be applied to all
blooms. Previous studies have shown that the amino acid L-lysine has
a strong inhibitory effect on the growth of the cyanobacterial genus
Microcystis (Kaya et al., 1996). Specifically, many studies have
focused on the effects of L-lysine on Microcystis aeruginosa,
one the most commonly appearing cyanotoxin-producing species.
Microcystis can cause rashes, burns, and blisters on the skin after
contact, as well as vomiting, nausea, headaches, diarrhea,
pneumonia, and fever when ingested (Le, 2009). L-lysine is a
promising solution to the problem of controlling harmful algae
blooms, as it is naturally occurring and safe for humans. More
recent studies have revealed that L-lysine has the ability to
eliminate M. aeruginosa cells specifically by inducing
oxidative stress on the cells, breaking down the cell membrane, and
infiltrating the photosynthetic system (Tian et al., 2018). In
another study, it was found that lysine could completely eliminate
certain species of Microcystis (Hehmann et al., 2002). Furthermore,
in a tank experiment examining the effects of lysine and malonic
acid on M. aeruginosa, it was found that the lysine killed
the algal cells, allowing the environment to recover and the aquatic
plants to grow (Kunimitsu et al., 2005). Lysine could prove to be a
valuable method in selectively controlling the growth of
Microcystis.
However, little research has been
done on the effects of L-lysine on other freshwater organisms and
the environment as a whole. This information is crucial in
determining if L-lysine could be a sustainable way to mitigate the
growth of Microcystis blooms. If lysine could maintain the condition
of an aquatic ecosystem without inducing any negative effects, then
it could be used to control future, real-world blooms of
Microcystis. In order to gain a better understanding of the
interaction between lysine and a freshwater ecosystem, this study
will use zooplankton species D. pulex to model the effects of lysine
on the aquatic environment. Zooplankton plays an important role in
most aquatic ecosystems because many larger organisms in higher
trophic levels rely on it as a food source (Lomartire, 2021). That
being said, it can be generalized that a decrease in the zooplankton
population would indicate a negative impact on the environment as
whole, since a change in food supply would affect the transfer of
energy within the ecosystem (Mooji et al., 2010). Meanwhile, a
drastic increase in the zooplankton population could cause
overpopulation in the environment.
Procedure
Culturing: M. aeruginosa UTEX LB 2385 was cultured in Bold 3N medium
under a 12hr light: 12hr dark cycle (Tian et al., 2018). Using a
hemocytometer to estimate population size, the algae was subcultured
in B3N medium prior to testing. D. pulex containers were
opened immediately upon arrival, with the cap resting on top to
allow air exchange. For five days of the week, the zooplankton were
fed a few drops of a yeast solution. Additionally, D. pulex
were kept under a 12 h light: 12 h dark cycle, with half the water
in the being replaced each week.
Experimentation:
D. pulex was opened immediately upon arrival, with the
zooplankton being randomly distributed into groups of five in a
6-well plate. The zooplankton were counted and separated into wells
until 3 plates were filled to obtain 18 total wells, containing 5
Daphnia each. The Daphnia were then separated into 6 test groups: 5
mg/L lysine, 8 mg/L lysine, 5mg/L lysine and M. aeruginosa, 8
mg/L lysine and M. aeruginosa, just M. aeruginosa, and a
control with no added substances. For test groups containing lysine,
5 or 8 microliters of a lysine solution containing 10 mg lysine per
1 mL dH2O were added to the 10mL of spring water in the test group.
For test groups containing M. aeruginosa, 1 mL of a
preexisting subculture was added to the 9mL of spring water
containing the zooplankton. Additionally, a control groups
containing just D. pulex and spring water was included to
account for any potential effects of the spring water or light
intensity on the zooplankton. For three days, the zooplankton in
each test group would be counted and recorded. Using the results of
the total death caused by lysine over the three-day period would
allow for a LC50 to be calculated by graphing the results. A test
containing M. aeruginosa and L-lysine was also included to
confirm the effects of lysine documented in previous studies. In a
6-well plate, 9 mL of B3N medium and 1 mL of an M. aeruginosa
culture were added to each well. Using a hemocytometer, the initial
population for each well was estimated to be 5*10^5 cells/mL. 5 and
8 microliters of a lysine solution containing 10 mg lysine per 1 mL
dH2O were added to the test groups, respectively.
Data
Collection A hemocytometer and compound light microscope
were used to estimate the algal population by taking the average
cell count between sixteen of the corner squares and multiplying by
a factor of 10^4. Completing this process yields an average cells/mL
count. This process was repeated over the course of three days, and
results were used to create a growth curve for the M.
aeruginosa population. Changes in the zooplankton population were
observed by physically counting the number of remaining zooplankton
in each well. The number of living cells in each well were added to
get a total population per test group.
A graphical abstract of the procedure, where the two concentrations
of lysine were added to D. pulex, M. aeruginosa,
or both
Figure 1
Figure 2
Figure 3
Figure 4
A picture of the actual experiment. Here,
all of the test groups containing zooplankton can be seen.
Analysis
Controls and M. aeruginosa Only: As seen in Figure
1, the average D. pulex population among the 3 groups
containing 5*10^5 cells/mL M. aeruginosa was 0.33, with only
one of the original fifteen organisms surviving the 48-hour period.
In other words, it can be said that the survival rate of the group
containing onlny M. aeruginosa was 6.67%. Meanwhile, the control
group containing only D. pulex experienced a net growth of
6.67%, with a count of 16 living organisms after the 48-hour period.
Lysine and D. pulex: Seen in
Figure 2, it was found that with both concentrations of lysine
alone, the zooplankton had a 67% survival rate, with a total of ten
from each group surviving the 48-hour period.
Lysine
and D. pulex in an HAB Setting: With 8 mg/L lysine
and M. aeruginosa, the zooplankton had a 53% survival rate
(Figure 2), which is significantly larger than the 6.67% zooplankton
survival rate for groups with only M. aeruginosa (Figure 1).
Meanwhile, D. pulex in the 5 mg/L concentration of lysine
with M. aeruginosa had an average population of 1.33 living
cells after 48 hours, or a 33% survival rate.
Lysine and M. aeruginosa: Using a hemocytometer,
populations for groups of M. aeruginosa containing 5 and 8
mg/L lysine were estimated each day. It was found that population
size began to decrease by the 48-hour mark when lysine was present
(Figure 3). The group containing 5 mg/L lysine decreased to a
density of 2.9 cells/mL, and a concentration of 8 mg/L lysine caused
the cell density to decrease to about 2.7 cells/mL. Meanwhile, the
control group had a final estimated population of 7.1 cells/mL.
While the decrease in cell density was not as dramatic as noted in
other studies (Tian et al, 2018), the presence of lysine still
caused a decrease in cell count, indicating it was responsible for
causing cell death.
Discussion/Conclusion
Lysine and M. aeruginosa: Although previous tests
had already documented and established the effects of lysine on M.
aeruginosa (Tian et al., 2018), this study included a test to
confirm the effects of lysine and act as a control if the results
from testing with D. pulex did not come out as anticipated.
In this experiment, the 5 mg/L and 8 mg/L lysine concentrations were
tested, as these were shown to cause the largest decrease in cell
density (Tian et al., 2018). While the results from this experiment
(Figure 3) were not as dramatic as expected, the lysine was still
shown to have a negative effect on the growth of M.
aeruginosa, indicating that it was responsible for cell death
within the community, thus confirming the effects of lysine on M.
aeruginosa.
Lysine and D. pulex:
To gain a better understanding of any potential ecological
consequences of using L-lysine to control HABs, tests examining the
effects of lysine on the D. pulex population were carried out
to see if it would have a significant effect on the marine food
chain. As lysine would only be used in the context of an HAB, the
experiment was split into two distinct test groups: a lysine-only
test, and a test containing lysine and M. aeruginosa, both of
which were split into test groups containing 5 mg/L and 8 mg/L
lysine. In the lysine-only group, it was found that both test groups
had a 66% survival rate at the P<0.05 significance level
(Figure 2). This does indicate that the lysine may have been
responsible for some cell death, especially when compared with the
control group, which experienced net growth of 6.67% (Figure 1).
However, the survival rate was still above 50%, indicating that the
population would still be able to grow in the presence of lysine.
Lysine on D. pulex undergoing a bloom of M.
aeruginosa: In the test group containing lysine and M.
aeruginosa, it was found that the D. pulex in the 8 mg/L
group had a 53% survival rate, and those in the 5 mg/L group had a
survival rate of 33% (Figure 2), both at the P<0.01
significance level. When compared to the group containing only M.
aeruginosa, which had a survival rate of 6.67%, these results
indicate that lysine supported the growth of D. pulex in the
presence M. aeruginosa, supporting the initial hypothesis.
Furthermore, these results suggest that higher lysine concentrations
have a greater effect on the growth of D. pulex while
undergoing a bloom of M. aeruginosa, as the group with 8 mg/L
lysine had a higher survival rate than that of the 5 mg/L lysine
group. This conclusion is consistent with the evidence that higher
concentrations of lysine will cause a greater rate of cell death
within the M. aeruginosa community (Tian et al., 2018), since
M. aeruginosa was shown to harm D. pulex (Figure 1).
It is important to note that these results do not necessarily
suggest that lysine directly promoted the growth of D. pulex
in the presence of M. aeruginosa, but rather indirectly
helped maintain the population of D. pulex by reducing the M.
aeruginosa population.
Future Research:
These results are only one step in showing that lysine may be a
sustainable method to control harmful blooms of Microcystis, so more
tests, such as in-situ tests, should be carried out to determine any
potential negative effects of L-lysine before it can be implemented
in real-world situations. Additionally, cost models and plans for
administering lysine could be constructed to scope the feasibility
of lysine as a method to control Microcystis blooms.
References
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