The Effect of Temperature on the Toxicity of Planktothrix Agardhii

THE EFFECT OF TEMPERATURE ON THE TOXICITY OF PLANKTOTHRIX AGARDHII

Christina Moore

A Thesis

Submitted to the Graduate College of Bowling Green State University in partial fulfillment of the requirements for the degree of

MASTER OF SCIENCE

May 2020

Committee:

George Bullerjahn, Advisor

Timothy Davis

Robert Michael McKay

Christina Moore

ABSTRACT

George Bullerjahn, Advisor

The western basin of Lake Erie has experienced issues with cyanobacterial harmful algal blooms

(cHABs) since the 1960s due to various factors over the decades. This resulted in a service shutdown in

Northwest Ohio in August of 2014, leaving at least 400,000 people without drinking water for three days. Since then, research has been conducted to learn about two of the cyanobacterial species that reside in western Lake

Erie. One of these species is Planktothrix agardhii, a filamentous cyanobacteria species that resides in

Sandusky Bay due to nitrogen depletion in the bay. While previous work has looked at the physical and

physiological factors that influence the growth of Planktothrix and other cyanobacteria, there are only a few that have looked at factors that drive microcystin production, a toxin found in Plantktothrix, and why these toxins have been produced in the first place. These papers have different ideas as to the purpose of these toxins. A strain of Planktothrix agardhii was grown and acclimated in a growth chamber with ten different temperatures to see how temperature affected the toxicity of the cultures. This involved looking at the cell density, toxin quota, and toxin quota per cell of samples collected during the experiment. Results show that the cell densities all increased at the end of the experiment and that 25.9°C had the highest cell density, while toxin quota was

higher at lower temperatures. These results support the hypothesis that lower growth temperature increases the

toxin centent of Planktothrix agardhii cells. There may also be a relationship between filament density,

microcystin content, toxin per filament ratio, and size of filaments. However, more work would need to be done

to assess that relationship. There also needs to an improvement in the method used to look at toxin content to

avoid an introduction in microcystin content errors due to low cell densities. This would involve looking at both

total and dissolved microcystins. iv

ACKNOWLEDGMENTS

I’d like to thank first and foremost my advisor, Dr. Bullerjahn, and the members of my

committee, Dr. Davis and Dr. McKay, for their wisdom and guidance through my entire

experience as a graduate student. I very much enjoyed the conversations I got to have with each

of you and for the laughs we all shared along the way. Thank you to Dr. Reiner for sending, to

the Bullerjahn lab, the Planktothrix agardhii strains that were cultivated and used for this

experiment. I’d also like to thank the other members of Dr. Bullerjahn and Dr. Davis’s lab,

specifically Emily Beers, for their advice and support throughout the experiment. Thank you also

to Michelle Neudeck, for keeping my chin and spirits up with your pep talks and for keeping my

sanity in check throughout my time here. I will deeply miss the opportunity to have

conversations with you in the office.

I’d like to also thank my parents, family, and friends for their unconditional love and

support as I worked through my blood, sweat, and tears to complete my degree. To my mom,

thank you for always being my support and for always telling me how proud you are. To my dad,

thank you for pushing me to find answers when necessary and for your support. To my sister,

Abigail, and to Alissa, thank you both for keeping me in line and for the distractions provided from our nights spent together. To the family dog, Louie, thank you for always being open for snuggles and for being your goofy self.

Last, but certainly not least, my biggest thank you goes to Bowling Green State

University. None of this would have been possible without all the opportunities and lessons I

experienced throughout my time as an undergraduate and a graduate. I am forever grateful to the

place I called home for the last six years. v

TABLE OF CONTENTS

Page

INTRODUCTION ...... 1

METHODS ...... 6

Cell Culture ...... 6

Growth Chamber and Acclimation Period ...... 6

Experimental Run ...... 7

Assessing Growth of Strains and Toxin Quota ...... 8

RESULTS ...... 10

Cell Growth ...... 10

Toxin Quota ...... 11

Toxin Quota per Cell mL-1 Ratio ...... 11

DISCUSSION ...... 13

REFERENCES ...... 16

APPENDIX A. FIGURES ...... 24

INTRODUCTION

Cyanobacteria are prokaryotes that have the ability to perform plant-type photosynthesis for energy (Whitton and Potts, 2012). Earliest records show that they have been around for approximately 3.5 billion years (Schopf 1993, 2006; Schopf et al. 2007; DeGregorio et al. 2009) and were the main drivers of oxygenation of Earth’s atmosphere during the Great Oxidation

Event (Schirrmeister et al. 2015). They have also been found to be the primary nitrogen fixers in the oceans (Diez et al. 2008). Cyanobacteria also produce accessory bile pigments that serve to harvest light for photosynthesis; a blue pigment called phycocyanin and in some species, a red pigment, phycoerythrin (Jaiswal et al, 2018). Despite being important to the environment, some species of cyanobacteria are harmful to freshwater ecosystems as they are responsible for formation of cyanobacterial harmful algal blooms, or cHABs (Paerl and Huisman, 2009;

O'Neil et al. 2012; Michalak et al. 2013; Visser et al. 2016; Berry et al. 2017). These blooms have been found in the lower great lakes and have been observed to produce microcystin, a liver toxin that is dangerous when consumed by humans and animals (Bullerjahn et al. 2015).

In the United States, the five Laurentian Great Lakes are an important source of freshwater for U.S. and Canadian citizens that live near these lakes (Steffen et al. 2014). Since the 1960s, Lake Erie is one of the most affected by cHABs, specifically in western Lake Erie.

(Smith et al. 2015; Bullerjahn et al. 2016). There have been several known causes of cHABs, with the main causes being eutrophication due mainly to increased agricultural nutrient loads of nitrogen and phosphorus, climate change and increased precipitation, and zebra mussel invasion

(Smith et al. 2015; Bullerjahn et al. 2016). Whereas Lake Erie cHABs in the 1960s and 70s were dominated primarily by nitrogen-fixing cyanobacteria, the resurgence of blooms in the 1990s saw the emergence on non-nitrogen fixing genera such as Microcystis and Planktothrix (Steffen 2

et al. 2014; Bullerjahn et al. 2016). In August of 2014, an elevated concentration of microcystin toxin, produced by cyanobacteria that was detected in finished drinking water, resulted in a service shutdown in Northwest Ohio, leaving about 400,000 residents without drinking water

(Smith et al. 2015). This event resulted in an increase of research done on the HABs in Lake

Erie. Today’s research on HABs have resulted in knowledge on the two main bloom-forming cyanobacteria genera in western Lake Erie. One of them is Microcystis spp., which dominates cHABs in the open waters of western Lake Erie (Brittain et al. 2000). The other is the primary focus of this research and is known as Planktothrix agardhii, which tends to dominate cHABs in the tributaries such as the Maumee River (McKay et al. 2018) and Sandusky Bay (Kutovaya et al. 2012; Davis et al. 2015; Hampel et al. 2019).

Planktothrix agardhii is a filamentous cyanobacterium that is one of the two most

common Planktothrix species that have been characterized and isolated with the other being

Planktothrix rubescens (Kurmayer et al. 2016). They are efficient light harvesters, meaning they

prefer areas of low light intensity and can be photoinhibited in high light (Mur et al, 1978; Van

Liere et al. 1979; Kurmayer et al. 2016). P agardhii prefers shallow lakes in temperate climatic

zones and in some sub-tropical regions (Suda et al. 2002; Kurmayer et al. 2016). They have been

found in fresh waters in the Northern Hemisphere and in some areas of Australia, New Zealand,

South America, and Morocco (Pridmore and Etheredge 1987; Baker and Humpage 1994; Kruk et

al. 2002; Bouchamma et al. 2004; Kurmayer et al. 2016). Through research on P. agardhii, toxic

strains of the species have been found to produce microcystin toxins encoded in the mcy gene

cluster, a cluster of 9-10 genes that use non-ribosomal peptide synthetase (NRPS) to produce

these toxins (Kurmayer et al. 2016). Planktothrix was considered part of the Oscillatoria genus,

until 1988 when it became its own genus, which was confirmed through 16S rDNA sequencing 3

(Wilmotte and Herdman 2001; Suda et al. 2002; Komárek et al. 2014; Kurmayer et al. 2016).

Research with P. agardhii in Sandusky Bay in Lake Erie resulted in the discovery that this

species can tolerate long periods of nitrogen depletion, a common phenomenon occurring at sites

affected by sedimentary denitrification (Davis et al. 2015; Salk et al. 2018).

Previous work by Oberhaus et al. (2007) investigated both P. agardhii and P. rubescens

to see how the growth of both species responded to temperature, light intensity, and light quality.

Results of the experiment found that P. agardhii did best at 25°C (Oberhaus et al. 2007). They

also found temperature to be the most important parameter when looking at the dominance of the

two species (Oberhaus et al. 2007). This is because they observed that P. rubescens was a better competitor at 15°C than P. agardhii, and that P. agardhii was the better competitor at 25°C than

P. rubescens (Oberhaus et al. 2007). This also comes from their observations regarding how the

species reacted to different light qualities and intensities. They saw that P. rubescens preferred

low levels of green light and displayed strong photoinhibition at high light intensity levels

(Oberhaus et al, 2007). Meanwhile, P. agardhii didn’t show a strong preference toward any light

quality and wasn’t as sensitive to high light intensity levels (Oberhaus et al. 2007). The results on

what growth conditions the species favored were found to coincide with the habitat in which they

are normally found (Oberhaus et al. 2007). This means that P. rubescens favored cold waters,

while P. agardhii favored warmer waters.

Numerous papers have been published that have investigated the effects of different

environmental factors on the production of microcystins. In one study using Microcystis

aeruginosa, light and iron levels were found to have had the most influence on the level of

microcystin toxins were produced (Jiang et al. 2008). They also found that a specific

combination of concentrations of nitrate, phosphate, iron, light intensity, and temperature were 4

critical in rapidly increasing both the growth of Microcystis and the production of toxins (Jiang

et al. 2008). Another study that used a model strain of Microcystis aeruginosa, the production of toxins was more influenced by the environmental effects on cell division rather than the effects influencing the metabolic pathways of the toxins produced (Orr and Jones 1998). This showed there was potentially a direct relationship between how fast cells were dividing and how much toxins were produced, yielding a constant per-cell concentration of microcystin (Orr and Jones

2003). Another claimed that an increase in total phosphorus concentrations increased the concentration of microcystins in Microcystis in Lake Erie (Rinta-Kanto et al. 2009). One last study found that both Microcysis and Planktothrix can use several different nitrogen forms and stressed the importance of managing both nitrogen and phosphorus sources when looking at ways to controlling cHABs (Davis et al. 2015; Gobler et al. 2016; Chaffin et al. 2018).

In 2018, a separate study looked at how a strain of M. aeruginosa responded to different concentrations and species of nitrogen, and to different temperatures (Peng et al. 2018). They used RT-qPCR, metabolomics, and toxin profiling to assess the response to these parameters.

Results showed that even though there was slow growth at lower temperatures, there was a high toxin-to-cell quota at these lower temperatures suggesting that temperature may have a physiological effect on toxicity (Peng et al. 2018).

While there have been studies that have looked at toxin production in cyanobacteria, there are only a handful of papers that have assessed the cellular functions of the toxins produced. One of these studies wanted to see if oligopeptide production, the class of peptide microcystin toxins belong to, could be a possible mechanism against chytrid parasites (Rohrlack et al. 2013). The results of their paper found that oligopeptide production does serve as a defensive mechanism against chytrids, but it doesn’t exclude the other possible functions they 5

serve (Rohrlack et al. 2013). Because there were both ribosomal and nonribosomal oligopeptides

used for anitchytrid defense, oligopeptides weren’t originally produced to serve as anti-chytrid

agents, rather it was a mechanism that evolved over time (Rohrlack et al. 2013).

Another study used M. aeruginosa strains with phytoplanktivorous and omnivorous fish

and found that the strains increased microcystin production in the presence of the fish (Jang et al.

2004). Their findings support their hypothesis that M. aeruginosa increased their toxin

production as a defense against fish using physical and chemical cues (Jang et al. 2004).

Another study proposed that microcystin is produced to regulate the amount of free iron

(Fe2+) in cyanobacteria (Utkilen and Gjolme 1995). The study used M. aeruginosa strains and

looked at how different nutrients affected their growth. They found that iron concentrations in

the strains went down as toxin production levels increased at first (Utkilen and Gjolme 1995).

After growing the strains in a medium that would affect iron uptake, they observed that toxin-

producing strains had a more efficient iron uptake system than strains that didn’t produce toxins

and that the toxins could be acting as chelator that keeps levels of free Fe2+ in cell low (Utkilen

and Gjolme 1995).

Taking all of this into account, and recognizing the more extensive studies on Microcystis spp., in this thesis, I wanted to test the role of temperature in the production of microcystin toxins in P. agardhii, as prior work on Microcystis blooms indicated that toxin levels were higher in the

early summer, when bloom densities and temperatures were lower (Ninio et al. 2019). The

hypothesis is that growth at lower temperature increases the per cell toxicity of P. agardhii,

similar to what is seen in Microcystis spp.

METHODS

Cell Culture

Nine strains, seven toxin-producing and two non-toxin producing, were collected from

Sandusky Bay, isolated, and were genetically characterized in the lab of Prof. Rainer Kurmayer

(University of Innsbruck, Austria). Microcystin-producing strains were labeled #1025-1033, with the two non-toxin producing strains labeled #1026 and #1027. All strains were maintained in 125 ml Erlenmeyer flasks containing WC medium and gently shaken on a shaker table on a bench to keep them in suspension (Guillard 1972). All cultures were grown at 22.5°C under a light intensity of 8µM/m-2/s-1 with standard incandescent lights and a 24:0 L:D cycle. Aliquots from

each strain was used to verify toxin-production using a Microcystins-ADDA ELISA 96-well

plate test kit (Abraxis, Warminister, PA). Once verified, 10 ml of inoculum was introduced into

Erlenmeyer flasks containing 25 ml of WC media and grown to 500,000 filaments mL-1 as a

starter culture for the experiment. At the beginning of the acclimation period, one microcystin-

producing strain (1033) was chosen for further experimentation. This strain throughout the whole

experiment was cultured in flasks with JM media (Schlösser 1982) after being transferred from

WC media and sub-cultured as needed. The reason the cultures were transferred from WC to JM

media was because it was observed that the strains grew better in JM than in WC media.

Growth Chamber and Acclimation Period

A growth chamber was constructed using an aluminum manifold that can hold 30 tubes at

up to 10 temperatures as described by Watras and Chisholm (1982; see Figures 1 and 2).

Temperatures in the growth chamber ranged from 17-30°C under a light intensity of 10µM/m-2/s-

1 and a 12:12 L:D cycle. After a cell count of strain 1033, a volume of 1.4 liters with appropriate 7

measurements of JM media and 1033 so that the volume contained 5E+05 filaments mL-1 of

1033. Once made, thirty 50 mL falcon tubes were filled with well-mixed stock culture to ensure

uniform abundance within each tube with 45 mL of strain 1033 in JM media (approximately

5E+05 filaments mL-1) and placed into the growth chamber. Each temperature gradient was

replicated in triplicates. Tubes in the growth chamber were gently bubbled with hydrated air in

order to keep the cells in suspension and to minimize evaporation. The tubes were allowed to

grow until they reached stationary phrase in order to acclimate to each individual temperature.

During this acclimation phase, 1mL was collected from a random replicate from each

temperature row (A, B, or C) every 2-4 days for counting purposes. Samples for cell counts and

biovolume measurements were then preserved by the addition of 20µL of Lugols iodine. During

sampling, 1mL of JM media was added back in to feed the samples and restore the original

culture volume. Counts were recorded between samplings to assess growth of the samples.

Experimental Run

Once the samples reached the stationary phase during their acclimation period, strains

were sub-cultured. This was done by taking the triplicate tubes from each temperature and

combining them into a large flask. One mL was then taken for counts and normalization of the

initial inoculum and JM media was added to obtain 5E+06 filaments per mL in 140mL volume.

Once the biomass and volume was achieved, 45mL was transferred into sterile Falcon tubes and

returned to the growth chamber. This was repeated for each temperature gradient. When all 10

temperature gradients were completed, 1mL was taken from each tube for an initial count. Every

other day after inoculation, sampling was conducted on all thirty tubes. During sampling, 1mL

was taken for counts and 1mL was taken for toxin analysis. 2mL of JM media was then added

back in. Tubes used for counting were preserved with 20µL of Lugols iodine. For toxin analysis, 8

each 1mL tube was centrifuged at 8000 rpm for 12 minutes at 15°C to pellet the biomass. The

supernatant was gently removed and discarded so only the pellet of algae remained in the tube.

Distilled water was then added back into tubes to replace media removed. Tubes were then

centrifuged at 2000 rpm for 30 seconds at 20°C to assure that the cyanobacteria were still present

in the tubes. All tubes were then stored in a -80°C freezer until the appropriate time to perform

ELISA tests.

Assessing Growth of Strains and Toxin Quota

Growth of the strains were assessed by measuring biovolume and chlorophyll concentrations of the strains in the chambers. To measure the biovolume, samples were concentrated via gravity and then enumerated (McKay et al, 2018). All samples were enumerated using microscopy to count the number of filaments present. The filament density was calculated by taking each filament count, multiplying it by 83.3, the observed average cell density per filament, and then entering that number into a template that calculated the cell density per mL.

Toxin quota results were collected by performing Microcystins-ADDA ELISA tests using

Abraxis ELISA 96 well plate kits (Warminster, PA). These tests were performed on toxin sample tubes from Days 6, 10, 14, 24, and 30. Sample tubes underwent a process of thawing the tubes completely and then freezing the tubes three times in order to lyse the cells. After the third freeze/thaw cycle, an aliquot was taken directly in order to enhance the sensitivity of microcystin according to the methods used by a study done by Fischer (2001; Davis et al. 2015). The ELISA tests were then run and evaluated as according to the manufacturer’s instructions. The content was measured in ug/L by calculating %B/B0 for each standard and then creating a curve on the y-

axis versus the microcystin concentrations on the logarithmic x-axis as instructed in the Abraxis kit manual. This standard curve was then used to find the results of the microcystin content of the 9 samples. One kit was used for each day and then the microcystin contents shown were taken to calculate the averages for each temperature, which is shown in Figure 5.

RESULTS

Cell Growth

Figure 3 shows average for all temperature, while Figure 4 shows the growth for the lowest and highest temperature, and the temperature that showed the best growth. Figure 4 was

made for purposes that most cultures showed similar growth during the experiment. All of the

cultures showed variability at the beginning cell density with the highest count at 7.43E+05 mL-1

and the lowest at 3.39E+05 mL-1 (Figure 3). Most of the cultures started to increase in growth

after day 14 (Figure 3). The highest cell density at the end of the experiment was observed at

25.9°C with a filament density at 8.05E+06 per mL with an exponential growth rate of 0.00387

cells per hour (Figure 4). The slowest growing culture was seen at 18.6°C as its cell density only doubled from 5.15E+05 per mL to 1.18E+06 per mL in 30 days with an exponential growth rate of 0.001152 cells per hour (Figure 3). The cultures at 30.1°C also showed a low growth rate in the beginning as it had the lowest filament density at the start of the experiment at 3.39E+05 per mL (Figure 4). During the first 18 days, the cultures showed a decrease in growth as it was at a density of 2.16E+05 per mL with an exponential growth rate of -0.001043 cells per hour (Figure

4). However, it slowly started to show growth afterward, and greatly increased from 1.68E+06 per mL at day 26, to 7.14E+06 per mL at day 28 with an exponential growth rate during that time of 0.030603 cells per hour (Figure 4). While most cultures showed a cycling up and down fluctuation in cell density throughout the experiment, all strains showed an increase in filament density from their starting count (Figure 3). 11

Toxin Quota

Most of the temperatures showed little to no increase in the microcystin quota during the

experiment. Lower temperature incubations (17.1 and 18.6°C) yielded the highest final

microcystin quota. Despite the fact that the culture incubated at 18.6°C grew very poorly (Figure

3), the culture continued to accumulate microcystins to yield a final quota matching the 17.1°C culture. The microcystin quota from day 6 to day 10 in 17.1°C increased from 0.5 ug/L to 7.21

ug/L before decreasing to 1.93 ug/L at day 14 and ending at 4.87 ug/L (Figure 5). 18.6°C

microcystin quota increased from 0.22 ug/L at day 6 to 4.78 ug/L at day 30 (Figure 5). All the

cultures at other temperatures didn’t even reach 1 ppb during the experiment according to the

results. However, 23.1°C showed some increase in microcystin as it increased from 0.23 ug/L at

day 6 to 0.83 ug/L at day 10 before decreasing to 0.37 ug/L by day 30 (Figure 5).

Toxin Quota per Cell mL-1 Ratio

Calculations were achieved by dividing the toxin quota by the cell density. Results for

18.6°C were excluded from these results due to poor cell growth during the experiment (Refer

back to Figure 3). Figure 4 shows results for all temperature gradients from Day 6 to Day 30.

Figure 5 shows the same results only from Day 14 to Day 30 for scaling reasons, due to the high

toxin content detected in the 17.1°C culture.

Much like with Figure 3, the toxin quota per cell mL-1 at 17.1°C greatly increased from

1.51E+03 femtograms (fg) per filament at day 6 to 1.69E+04 fg per filament at day 10 before decreasing to 9.16E+02 fg per filament at day 30 (Figure 6).The cultures at 30.1°C showed the best toxin quota per cell mL-1 at day 6 with 2.91E+03 fg per filament (Figure 6). However, it

decreased to 2.10E+01 fg per filament at day 30, having one of the lowest toxin quota per cell 12

mL-1 by the end (Figure 6). Between days 14 and 30, 23.1°C showed the next best toxin quota

per cell mL-1 even though it decreased from 7.16E+02 fg per filament at day 14 to 1.25E+02 fg per filament at day 30 (Figure 7). 17.1°C still showed the highest toxin quota per cell mL-1 numbers between days 14 and 30, although the numbers decrease from 1.56E+03 fg per filament at day 14 to 9.16E+02 fg per filament at day 30 (Figure 7). Those numbers are much higher than the other temperatures in that time frame as they were reported in the 1E+01 and 1E+02 fg per filament ranges between days 14 and 30 (Figure 7). These data show that the toxin quota per cell mL-1 was maximized at the lowest growth temperatures.

DISCUSSION

When the experiment first started, it seemed as if the results and predictions made were

going to be based on the literature that was published looking into P. agardhii and the factors

that influenced its growth and toxicity. Based on the results shown above, there are a few key observations to point out.

First, when looking at Figures 3 and 4 for the cell density, there is an increase in biomass by the end of the experiment at all temperatures. However, the samples at 25.6°C showed the

highest cell density at the end on day 30 as both figures show. This is similar to what was

reported by Oberhaus and colleagues (2007). The results reported showed that P. agardhii had a

higher growth rate and competed better against P. rubescens at 25°C than at 15°C (Oberhaus et

al. 2007). However, Figure 3 shows that 17.1°C grew well and yielded higher biomass than

30.1°C, 24.3°C, 27.1°C, 20.2°C, and 18.6°C. On the other hand, Figure 3 also showed that 25.9°C,

23.1°C, 21.7°C, 28.9°C had a higher biomass than 17.1°C.

Results from the toxin analysis show that 17.1°C and 18.6°C increased in toxin quota

while all other temperature incubations showed low increases or a slight decrease in toxin quota.

Notably, the 17.1°C culture on day 10 yielded a very high microcystin quota on day 10 at 7.21

ug/L, which is unusual considering the cultures for that temperature had not yet begun growing

at high rates. It’s possible that there may have been a slight error during the ELISA assay for that

run as one of the triplicates showed a toxicity of 17.21 ug/L while the other two triplicates were

2.89 and 0.97 ug/L. However, the rest of the numbers from 17.1°C show a steady increase,

therefore this observation may not be concerning. 14

Walls et al. (2018) found that P agardhii bloom that dominated a eutrophic freshwater

lake showed elevated extracellular microcystin concentrations between 20-25°C both in the lake

and in lab cultures. They found that the release of microcystins from cells in culture sharply

decreased around 30°C (Walls et al. 2018). This also would explain why samples between 25.9-

30.1°C showed a decrease in toxin-production by the end of the experiment. In general, the

results reported in the paper are in general agreement to this study, showing that intracellular

toxins are lower at higher temperatures.

Last, figure 6 shows that all temperature gradients decreased in toxin quota per cell mL-1

by the end of the experiment. 17.1°C did show an increase in toxin quota per cell mL-1 from day

6 to day 10, but this was more than likely due to high increase in microcystin as the filament

density had not increased that much between those days. Figure 7 also shows the toxin quota per

cell mL-1 at 17.1°C was at least doubled that of the other cultures grown at higher temperatures.

Therefore, Figure 7 clearly shows that the temperature did have a significant effect on the

toxicity of the cultures. Nonetheless, future work should focus on relationships between filament

density, microcystin content, toxicity per filament, and filament size and seeing if there are

additional interactions among all four parameters. Regardless, these results do support the

hypothesis that the growth temperature P. agardhii grows in has a significant effect on its toxicity.

One technical aspect of the work can be improved to yield more accurate toxin data at lower cell densities. In the current study, 1 mL aliquots of cells were centrifuged, pelleted, and stored at -80°C prior to the ELISA assay. Given that low densities of cells from early time points

in the growth curve may not be quantitatively recovered by this method, errors in microcystin

content may be introduced. This could explain the high and variable microcystin values detected 15 at day 10 in the 17.1°C culture (Figures 5 and 6). Future experiments may simply assay cell suspensions without centrifugation, and these samples analyzed for total and dissolved microcystins by testing both the cell suspension and culture filtrates.

Potential steps moving forward include repeating this experiment, but this time looking at what genes were being transcribed and the quantitative content of these genes. Perhaps looking at the genes being transcribed could help explain more how temperature plays a role in affecting the toxicity of P. agardhii. Another possible step could be to repeat this experiment, but have the experimental run go for longer than 30 days to see if there’s any different findings by extending the run to the cultures reaching stationary phase. One last suggestion to point out would be to perform statistical tests that would strengthen the data. The results of this experiment don’t include any results of statistical analysis, which makes it difficult to truly support the findings of this experiment. Going back and performing the appropriate statistical analysis tests on the data could help support the results and tell me which data are significant.

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APPENDIX A. FIGURES

Figure 1. Photograph of the temperature gradient incubator. This is what one sees from the

outside of the incubator. One the side is a timer that controls the lighting inside the

incubator. There are also coolers on the bottom to help regulate the colder temperature

gradients. There were also air pumps on each side of the incubator to filter the air going

into the tubes. 25

Figure 2. Photograph of the inside of the temperature gradient incubator. One can see the array of

triplicate sample tubes exposed to the temperatures 17-30°C. There are two aluminum

manifolds to control the temperatures and flasks to allow filtered and hydrated air to

reach the tubes. Each set of tubes has a needle valve to control overall air pressure. Each

individual Falcon tube has a pinch valve that regulates bubbling. 26

Average Cell Density per mL 1.00E+07

8.00E+06

6.00E+06

4.00E+06

2.00E+06 Cell Density/mL Cell

0.00E+00 -5 0 5 10 15 20 25 30 35 17.1°C 20.2°C 21.7°C 23.1°C 24.3°C -2.00E+06 Day 25.9°C 27.1°C 28.9°C 30.1°C

Figure 3. Growth as determined by average cell density per mL over the 30 day course of the

experiments. These were collected every other day from day 0 to day 30 except Day 16. 27

Average Cell Density (per mL) for 17.1°C, 25.9°C, and 30.1°C 1.00E+07

8.00E+06

6.00E+06

4.00E+06

2.00E+06 Cell per mL per Cell 0.00E+00 -5 0 5 10 15 20 25 30 35 -2.00E+06 Days

17.1°C 30.1°C 25.9°C

Figure 4. Average cell density per mL for 17.1, 25.9, and 30.1°C. Growth curves for the 17.1,

25.9, and 30.1°C triplicate samples. The same results from Figure 1 are shown, except

that they show the highest temperature, the lowest temperature, and the temperature that

yielded the highest ending cell density. 28

Average Microcystin Quota (ug/L) 10 )

0 0 5 10 15 20 25 30 35 40 -2 Microcystin Quota(ug/L Microcystin Day

17.1°C 18.6°C 20.2°C 21.7°C 23.1°C 24.3°C 25.9°C 27.1°C 28.9°C 30.1°C

Figure 5. Average microcystin quota (ug/L). These show the average results of the ELISA assay

of samples taken on days 6, 10, 14, 24, and 30. Results of each day come from the

standard curve of the standards from the kit used on the samples from the specific day.

Average Microcystin Quota (ug/L) per Cell per mL 2.50E-05

2.00E-05

1.50E-05

1.00E-05

5.00E-06

0.00E+00 0 5 10 15 20 25 30 35 40 -5.00E-06 Microcystin (ug/L) quota Microcystin Day

17.1°C 20.2°C 21.7°C 23.1°C 24.3°C 25.9°C 27.1°C 28.9°C 30.1°C

Figure 6. Average microcystin quota (ug/L) per cell per mL. (Excluding 18.6°C). These were the

average taken for days 6, 10, 14, 24, and 30 after dividing the microcystin content by

filament. The higher the number, the more toxin each individual filament contained. 18.6° was excluded due to its poor growth over the experiment. 29

Average Microcystin Quota (ug/L) per Cell per mL 2.00E-06

1.50E-06

1.00E-06

5.00E-07 Microcystin (ug/L) Microcystin

0.00E+00 0 5 10 15 20 25 30 35 40

-5.00E-07 Day

17.1°C 20.2°C 21.7°C 23.1°C 24.3°C 25.9°C 27.1°C 28.9°C 30.1°C

Figure 7. Average microcystin quota (ug/L) per cell per mL from day 14 to day 30 (Excluding

18.6°C). The same results from figure 4 are shown up close and only from day 14 to day

30 for scaling reasons that had to do with high toxin content that was found in 17.1°C on

day 10.