APEC Youth Scientist Journal Vol.7 / No.2
A POTENTIAL CAUSE OF ALGAL BLOOM: ANTIBIOTICS’
EFFECT ON EUKARYOTIC CHLOROPLASTS OF CHLORELLA
VULGARIS

Jai Eun HUH1
1
Seoul International School, 15, Seongnam-daero 1518beon-gil, Sujeong-gu, Seongnam-si,
Gyeonggi-do 461-830, Republic of Korea
ABSTRACT
Antibiotics primarily inhibit the growth of prokaryotic cells. The theory of
Endosymbiosis states that eukaryotic chloroplasts and mitochondria originate from the
prokaryotes, and recent oncologist research reveals that antibiotics can directly exert
influence on eukaryotic mitochondria. Similarly, this research probes at the possibility of
antibiotics specifically targeting eukaryotic chloroplasts of green alga, Chlorella vulgaris, in
an attempt to configure the mechanism through which antibiotics often affect algae. The
other half of the research explores the residual environmental risks associated with the
presence of antibiotics in freshwater ecosystems, and if antibiotics can possibly be a direct
cause of algal blooms. Well over half of the antibiotics, not metabolized and culled out by the
sewage plant, end up near the top of the water column: consequently, algae on the surface
water are exposed to these antibiotics. Excessive algal growths deplete dissolved oxygen (DO)
level and compromise water quality; specifically one of their types, harmful algal blooms
(HABs) are disastrous to the aquatic biota. Many obvious and common factors – excessive
nutrients and lack of water circulation - are known to encourage these algal blooms.
However, as antibiotics in low dosage can stimulate bacterial resistance and encourage
growth, the experiments showed that antibiotics could foster algal growth and be a cause of
algal blooms.
Key words ; algal bloom, Chlorella vulgaris, chloroplast, antibiotics, antibiotic effect

Correspondence to : Jai Eun HUH ([email protected])
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1. INTRODUCTION
Antibiotics are prescribed to affect prokaryotic cells such as bacteria. Each type of
antibiotics affects specific target mechanism such as the cell wall or cell membrane synthesis,
signaling of DNA replication, DNA transcription, RNA translation, or protein synthesis.
Whilst antibiotics are designed to work against bacteria, antibiotics of low concentration can
increase bacterial resistivity and even stimulate growth. A recent trend in Oncology suggests
that the “side-effects” of some antibiotics that directly influenced eukaryotic mitochondria
can be utilized in chemotherapy (Lisanti et al., 2015).
Eukaryotic chloroplasts and mitochondria set themselves apart from other inner
organelles by having double cellular membranes and DNAs. According to the theory of
Endosymbiosis, chloroplasts and mitochondria have evolved from prokaryotes when bigger
cells engulfed them to be integrated into parts of eukaryotes. Similarly, this research proposes
a working mechanism that certain antibiotics directly influence eukaryotic algal chloroplasts.
Previously research has shown that certain types of commonly used antibiotics can
directly inhibit algal growth, as did Ciproflaxin, Gentamycin, Vancomycin even at low
concentrations with green alga P. Subcapitata (Magdelano et al., 2014). Additionally, by
eliminating prokaryotes from cultures of eukaryotic algae (Kviderova & Henley, 2005)
antibiotics can indirectly foster the algal growth removing the neighboring bacteria. On the
other hand, possibility of antibiotics directly encouraging algae growth has not been
contested in literature, as this research suggests.
Pharmaceutical compounds (Ph.Cs.) found in the environment pose extensive threats to
the local biota. A huge amount of antibiotics – in a year, United States of America alone,
3300 tons of antibiotics, were purchased (Alliance for the Prudent Use of Antibiotics, 2011)
and consumed. However, 30 – 90 % of the total human or animal administered antibiotics
are execrated in their active form (Rang et al., 1999) and end up in wastewater treatment
plants in which Ph.Cs. are not effectively filtered (World Health Organization, 1997). Hence,
these antibiotics float on the surface water (Halling-Sørensen et al., 1998) in low
concentrations but still in their active forms.
While the extent of effects of these antibiotics on ecosystems cannot be readily grasped,
this line of research calls attention to an environmental phenomenon, algal blooms. While
algae are our main producers of oxygen and a promising source of clean energy, their
excessive growth can compromise the overall aquatic environment by lowering DO level.
Perhaps, more alarmingly, Harmful Algal Blooms (HABs) that release toxins and degrade
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water quality continually plague the fresh bodies of water. In the Great Lakes, occurrences of
HABs have increased since the mid-1990s. Lake Erie experiences increasingly wider-scaled
explosive blooms every summer (Bridgeman et al., 2013). Coincidentally, in Lake Michigan,
antibiotics and other chemicals were discovered in more than half of the collected samples
(Source et al., 2012).
Scientists have condemned agricultural practices, increased intensity of precipitation,
and weak lake circulation as obvious factors contributing to massive algal blooms. However,
if the paper’s understanding of antibiotics’ effect on algal chloroplasts is validated through
varied in situ experiments, then antibiotics found in environment can also be a potent factor
of algal blooms. Algal blooms, however, are also heavily tied with temperatures. Lake
Paldang of Han River, Lake Daechung of Geum River System and downstream of Nakdong
River have increasingly reported incidences of HABs, and highest phytoplankton biomass
was shown between months of December and next March (Park et al., 2011). Lake Erie’s
algal blooms reach their peaks in early October, but in June Cyanobacteria, a major player in
the algal blooms was undetectable (Wynne et al., 2012). Algae in vitro are grown the best in
higher temperature: from the range of 25°C, 27°C, and 30°C, algal growth was maximized at
30°C (Cassidy, 2011). However, real-life algal blooms do not reflect the similar trend, and
there seem to be more intricate ties between temperature and real-life algal blooms.
Thus this research has three original areas of inquiry: 1) Mechanism through which
antibiotics affect algae 2) Antibiotics in low concentration fostering algal growth 3)
Temperatures’ specific ties to the effectivity of antibiotics on algae.
2. MATERIALS AND METHODOLOGY
2.1. Preparation of Algal solution and Measuring Absorbance
Two tablets of Chlorella Vulgaris were dissolved in 400 ml of distilled water using
magnetic stirrer to create algal mixture for each experiment. Then mixtures were kept in
conical tubes. 1ml of algal mixture was put into clear cuvettes and measured for overall
growth with 530 nm UV wavelength for overall growth; 645 nm, chlorophyll b; 663 nm,
chlorophyll a using UV Spectrophotometer. Mixtures were wrapped in aluminum foil to keep
the amount of light equal: One set to be stored in 5°C for 130 hours, the other set to be stored
in 5°C for the first 48 hours and in room temperature (RT) for the next 72 hours. At fixed
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time intervals, algal mixtures were checked for absorbance and compared with day 0
absorbance to investigate if algal mixture can be stored in 5°C.
2.2. Algal Growth by Types and Concentration of Antibiotics
1ml of 1000x of Ampicillin, Chloramphenicol, Kanamycin, Gentamycin, Streptomycin,
and Cefazedone were diluted using aseptic techniques. 10µl of 0.1x of each antibiotic was
mixed with 1ml of algal mixture and put into conical tubes. Control was mixed with 10µl of
distilled water. After 48 hours, absorbance was measured and compared with day 0
absorbance. 10µl of 0.001x, 0.01x, 0.1x, 1x, 10x, 100x, 1000x of either Ampicillin,
Streptomycin was mixed 1ml of algal mixture. After 48 hours in RT, absorbance was
measured and compared with day 0 absorbance.
2.3. Algae Growth by Temperatures
10µl of either 0.1x Ampicillin or Streptomycin was injected into 1ml of algal mixture
prepare. Each set was put into 5°C, RT, and 30°C respectively, wrapped in aluminum foil to
keep amount of light consistent. The algal mixtures were checked for absorbance after 48
hours.
2.4. Effect of CaCl2 on algal growth
Two sets of 1ml of mixture were centrifuged at 4500 rpm for 6 minutes. Supernatant of
mixture was removed. Algal pellet was mixed with 500µl of 0.1M CaCl2 and cooled in ice
bath for 15 minutes. Again the mixtures were centrifuged. Supernatant of mixture was
removed. Pellet was mixed with 100µl of 0.1M CaCl2 and cooled in ice bath for 30 minutes.
For the control group, instead of 0.1M CaCl2, distilled water was added. Then 900µl of water
were added. After two days in RT, control group and CaCl2 group were checked for
absorbance. Calcium chloride did not affect proliferation of algae, but chlorophyll b level was
slightly lowered. Thus, calcium chloride could be used for the follow-up experiment.
2.5. Algal Growth after Heat-Shock Treatment
Mixtures underwent Heat-Shock Treatment. After final ice treatment, 900µl of either
Ampicillin or Streptomycin solutions were added to make 1ml of algal mixtures containing
10x ampicillin, 0.001x ampicillin, 1x streptomycin and 0.001x streptomycin respectively.
The control was mixed with 900 µl of distilled water. After two days in RT, the mixtures
were checked for absorbance.
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2.6. Algae Growth after Freezing Treatment
1ml of algal mixtures containing 10x ampicillin, 0.001x ampicillin, 1x streptomycin, or
0.001x streptomycin was cultured in RT for 48 hours. The absorbance was reset to 0.1 and
stored in -20 °C for 48 hours. The absorbance was reset to 0.1. After 48 hours in RT, the
absorbance was measured.
3. RESULTS AND DISCUSSION
3.1. Algal Storage in 5°C
To see if algae mixture could be preserved in 5°C for experiments, algae mixture were
placed first in 5°C and later in 5°C and RT. For algae mixture placed in 5°C for throughout,
between Day 0 and 1, the algae growth increased; Day 1 and 2, decreased sharply; Day 2 and
5, increased. Chlorophyll a and b showed a similar trend. However, chlorophyll a never
recovered from the initial sharp decrease between Day 1 and 2 and could not keep up with the
overall growth (Figure 1). During the first 24 hours, even under restricted supplies of
dissolved oxygen (DO), light etc., healthy cell division and regular photosynthesis respiration
could have taken place through light-independent reactions. During Day 1 and 2, however,
initial supplies of DO, COD, etc. necessary for cellular homeostasis and metabolic reactions
could have been depleted, causing the sharp decrease in all three levels. At this stage,
chloroplasts may have been damaged. Between Day 2 and 5, portion of algae could have
been damaged or died out; but portion that survived divided unhealthily and divided rapidly.
Thus, stress imposed by restrained environment may have triggered the overgrowth of algae.
If chloroplasts were negatively affected by light-restricted conditions in 5°C, algae
should have grown healthily when placed outside with some exposure to light. The follow-up
experiment placing stored algae outside showed otherwise. Like algal mixtures placed in 5 °C,
even when algae were placed outside, the amount of chlorophyll a and b did not recover from
the decrease, as growth absorption increased, but levels of chlorophyll a and b, indicators of
algal health, were negatively affected (Figure 2). In RT, the data showed a sharper increase
for overall growth, as higher temperature may have sped up enzyme activity and stimulated
algal growth (metabolic reaction happens most vigorously at higher temperatures). But finite
supply of oxygen, carbon dioxide, and other dissolved substances would have been depleted
at a faster rate, thus accelerating deterioration process of algae. Thus, it is predicted that some
algae must have died releasing toxins to set off chain reactions of other neighboring algae.
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Even though overall growth was increased, the level of chlorophyll a was negatively affected
and algae’s condition quickly deteriorated. Thus, it was clear that storing in 5°C must have
spurred unhealthy drop in chlorophyll levels. And the residual impact lasted even after the
algal mixtures were taken out to RT, as RT algal growth was excessive and short-lived.
Therefore, it was concluded that for every other follow up experiments algal mixtures
were to be freshly prepared and experiments would be restricted to the time frame of 48 hours.
3.2. Algal Growth by Types of Antibiotics
To see the growth of algae when treated with different types of antibiotics, algae were
cultivated with commonly used antibiotics such as Penicillin, Ampicillin, Chloramphenicol,
Kanamycin, Cefazedone, Gentamycin, and Streptomycin and measured for growth after 48
hours. For 530 nm, only the algae treated with Ampicillin had a higher absorbance level than
that of the control group. The algae treated with other antibiotics showed similar growth with
that of the control group, while the algae treated with Cefazedone had a noticeably reduced
growth (Figure 3).
Figure 2: Overall algal growth after
treating algae with different types of
antibiotics
Figure 1: Absorbance level for
chlorophyll a and b after administration
of different types of antibiotics
The pattern in absorbance level of chlorophyll a and b was similar to that of the overall
growth (Figure 4). Thus, certain type of antibiotics can have a very specific effect on algal
growth. Antibiotics that significantly affected green alga, Chlorella Vulgaris were Ampicillin
and Cefazedone and by nature are similar to β-lactam antibiotics in that they are designed to
influence bacteria externally (through cell wall); however, others designed to affect bacterial
inner organelles such as Kanamycin, Gentamycin, and Streptomycin only negligibly affected
algae (Figure 5).
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Algae administered with Streptomycin had the most similar chlorophyll mass and
overall growth to those of the control group. Therefore, for follow up experiments regarding
algal growth, Streptomycin and Ampicillin were used.
Figure 3: Predicted pathways for antibiotics on Chlorella vulgaris
3.3. Algal Growth by Concentrations of Antibiotics
To see if algae are more significantly affected depending on the concentrations of
antibiotics, algal mixture were treated with varied concentrations of Ampicillin or
Streptomycin and measured for absorbance. Small peaks were at 0.001x Streptomycin, 1x
Streptomycin, 0.001x Ampicillin, and 10x Ampicillin (Figure 6). Peaks were arbitrary and
thus largely discounted for further explanations.
Figure 4: Absorbance measures of overall growth by different concentrations of Ampicillin
or Streptomycin
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3.4. Algae Growth by Temperatures
To investigate at which temperature the antibiotics’ effect would be the most amplified,
algal mixture treated with antibiotics were placed in 5 °C, 30 °C and RT respectively. In 5 °C,
Ampicillin inhibited, but Streptomycin significantly encouraged overall growth of algae. In
30 °C, both antibiotics suppressed overall growth of algae. In RT, Ampicillin spurred, but
Streptomycin inhibited overall growth (Figure 7).
Figure 5: Overall absorption of algae based on temperatures
Temperature-specific pattern is shown similarly for levels of Chlorophyll a and
Chlorophyll b (Figure 8 & 9). Ampicillin promoted algal growth in RT, but in lower
temperature suppressed growth. Contrastingly, Streptomycin, at lower temperature promoted,
but in higher temperature suppressed growth. Thus, certain antibiotic’s effect on algae is
highly specific to the temperature – thus, in real-life, seasonal and regional factors play a
huge role in determining antibiotics’ behaviors. Furthermore, for antibiotics that foster
growth in lower temperatures like Streptomycin, can contribute to “winter blooms” or algal
growth in colder climates.
Figure 6: Absorbance of chlorophyll a
based on temperatures
Figure 7: Absorbance of chlorophyll b
based on temperatures
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3.5. Algal Growth after Heat-Shock Treatment
For 530 nm of UV wavelength, the control group had an absorbance of 0.828A and
CaCl2 group had an absorbance of 0.826A. The difference between the two groups is
negligible and thus, CaCl2 does not affect algal growth significantly. For chlorophyll a and b,
similar conclusion could be drawn (Figure 10). Thus, Heat-Shock Treatment could be used
for follow-up experiments.
Figure 8: Absorbance of algal mixtures treated with Calcium Chloride. Algae were treated
with 0.1M of CaCl2. The overall growth was checked with 530 nm; Chlorophyll a, 663 nm;
Chlorophyll b, 645 nm.
3.6. Antibiotics’ Effect on Algal Growth after Heat-Shock Treatment
To see if antibiotics specifically target algae’s inner system, algal mixtures went
through Heat-Shock Treatment to allow antibiotics to bypass algal cell wall and membrane,
and inner organelles were directly exposed to antibiotics. For 530 nm of UV wavelength,
0.797A, 0.770A, and 0.879A were recorded for the control, 0.001x Streptomycin, and 1x
Streptomycin group respectively. For chlorophyll a and b, 0.001x Streptomycin group had
larger absorbance than the control and 1x Streptomycin group had the largest absorbance
recorded (Figure 11). For 530 nm of UV wavelength, 0.797A, 0.815A, 0.883A were recorded
for the control, 0.001x Ampicillin, and 10x Ampicillin group respectively. For chlorophyll a
and b, the control had the lowest and 10x Ampicillin group had the highest absorbance
(Figure 12).
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Figure 9: Absorbance of algal mixtures
treated with 0.1 M CaCl2 and
administered differing concentrations of
Streptomycin. The overall growth was
checked with 530nm;
Chlorophyll a, 663nm; Chlorophyll b,
645nm
Figure 10: Absorbance of algal mixtures
treated with 0.1 M CaCl2 and
administered differing concentrations of
Ampicillin. The overall growth was
checked with 530nm; Chlorophyll a,
663nm; Chlorophyll b, 645nm
After Heat-Shock Treatment, for both Streptomycin and Ampicillin, the higher the
concentrations, the effect on algal growth was more pronounced. Without going through
Heat-Shock Treatment, 1x and 0.001x Streptomycin group only slightly suppressed algal
growth. But once Streptomycin was made to bypass the cell wall and cell membrane through
Heat-Shock Treatment, 1x Streptomycin group significantly induced growth. Similarly, 10x
Ampicillin group only negligibly affected algae. But once Ampicillin was administered after
CaCl2 treatment, 10x Ampicillin group significantly induced growth. Thus, it suggests that
antibiotics work directly on the inner organelles of algae.
3.7. Algal Growth after Freezing Method
To see if antibiotics specifically target chloroplasts, primarily chloroplasts were
ruptured through defrosting technique. For control, difference in overall absorptions of algal
mixture placed in RT and algal mixture placed in -20°C was significant. However, for
Ampicillin and Streptomycin, freezing sets had only slightly reduced values compared to the
ones placed in RT (Figure 13). On the other hand, if bacteria were treated with higher
concentration of antibiotics, bacteria would have died. But algae, when having been directly
exposed to higher concentrations of antibiotics, showed less severe response to freezing. Thus,
algae, when subjected to the initial stress from antibiotics, might have turned on self-defense
mechanism to prevent severe damage from freezing. Although for the control defrosting
eliminated most of algae, algal mixtures previously exposed to antibiotics were not affected
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by freezing as much. Algal inner organelles are affected by defrosting technique, but
chloroplasts, given their high free water content and abundance in algal cells, are the only
organelle that could have sustained algal growth and triggered the defense mechanism when
antibiotics were added.
Figure 11: Overall algal growths after freezing treatment for 530 nm UV wavelength
4. CONCLUSION
Green alga, Chlorella Vulgaris reacted sensitively to antibiotics when its inner
organelles were directly exposed or before it was defrosted. Thus, the study reveals that
certain antibiotics work on algae by directly affecting eukaryotic algal chloroplasts.
Usually algal blooms are a consequence of poor agricultural management, excessive
nutrient run-off, or weak water circulation; however, addition of residual antibiotics into the
ecosystem could make such episodes more frequent. Furthermore, understanding behaviors of
antibiotics toward algae specifically linked with temperatures may help improve and make
sense of the otherwise tenuous trend shown for peaks of algal blooms throughout the year.
However, for future experiments, experiments would have to be 1) conducted in situ, to
account for other environmental factors that may act interactively with antibiotics 2)
conducted with wider varieties of algae known to cause HABs 3) longitudinal to account for
long-term, residual effects of antibiotics on algae.
5. ACKNOWLEDGEMENTS
A special thanks to Sun Eui Kim, an executive director at Nature Science Institute, for
donating and supervising the use of UV Spectrophotometer, centrifuge, and general science
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equipment. Also thanks to Erik Anderson, Seoul International School Biology teacher for
suggesting this line of inquiry.
6. REFERENCES
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Jai Eun HUH
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