The Role of Nitrogen Availability on the Dominance of Planktothrix Agardhii in Sandusky Bay, Lake Erie

Home , Microcystis, Oscillatoriales, Planktothrix, Planktothrix agardhii

THE ROLE OF NITROGEN AVAILABILITY ON THE DOMINANCE OF PLANKTOTHRIX AGARDHII IN SANDUSKY BAY, LAKE ERIE

Daniel H. Peck

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

August 2020

Committee:

George Bullerjahn, Advisor

Timothy Davis

Robert McKay

Daniel H. Peck

George S. Bullerjahn, Advisor

Sandusky Bay and Lake Erie are plagued with harmful algal blooms every summer.

Sandusky Bay is a drowned river mouth that is very shallow and turbid and is dominated by

Planktothrix agardhii, while Lake Erie is dominated by Microcystis aeruginosa. Both species of cyanobacterium are non-diazotrophic and produce microcystin, a hepatotoxin. A competition experiment was conducted culturing both species alone and in coculture at nitrogen (nitrate) replete, nitrate restricted, and nitrogen-free environments. Planktothrix grew better alone at nitrogen restricted medium than in co-culture with Microcystis. In coculture, Microcystis was dominant over Planktothrix however, that dominance decreased as nitrogen was reduced in each treatment. In the nitrogen replete environment, the coculture produced significantly more toxin than the monocultures and in the no nitrogen environment the Planktothrix monoculture produced more toxin than the Microcystis monoculture or the coculture.

The community composition in Sandusky Bay was monitored over the winter and spring months (January-April) to see how it changed as time progressed. Nutrient amendment experiments were also conducted adding nitrate, phosphate, and a combination of nitrate and phosphate to stimulate growth and identify any possible nutrient limitations. The initial community yielded low cell densities until the temperature increased and cell abundances followed shortly thereafter. Planktothrix dominated over the winter followed by a transitional period of cryptomonad and diatom dominations before transitioning back to Planktothrix. Both nitrate and phosphate were limiting Planktothrix growth in the spring, while nitrate alone was limiting the overall community. iv

To my parents, David Peck and Sandra Maltzman for their constant support throughout

everything.

All the people I have met at BGSU and in the greater Bowling Green Community. I have truly

enjoyed my time in Ohio and am regretful to see it end. v ACKNOWLEDGMENTS

I would like to thank:

George S. Bullerjahn, Timothy Davis, and R. Michael McKay for giving me the opportunity to continue my education at the graduate level, for the many opportunities both in the lab and in the field for me to learn and grow, and for their advice, understanding and patience.

William Cody for assisting with the cellular enumeration for the sediment incubation portion of my thesis.

My undergraduate research advisor Dr. Raymond L. Kepner at Marist College for allowing me to conduct research with him and cultivating my love of research that has helped me throughout the entire journey.

The crews on the CCGS Limnos and CGC Neah Bay.

The entire BGSU Lab: Kaitlyn McKindles, Michelle Neudeck, Christina Moore, Kari

Lane Shupe, Jay DeMarco, Emily Beers, Callie Nauman, Seth Buchholz, Laura Reitz, Matthew

Kennedy Dr. Paul Matson, and honorary lab member/Dr. McKay’s postdoc Dr. Thijs Frenken. vi

TABLE OF CONTENTS

Page

CHAPTER 1: INTRODUCTION ...... 1

1.1: Shallow Nearshore Cyanobacterial Harmful Algal Bloom Environments ...... 1

1.2: Composition of Western Lake Erie cHABs ...... 2

1.3: Physicochemical Factors That Affect cHABs ...... 3

1.4: How Planktothrix Acclimates to Low Nitrogen Environments ...... 4

1.5: Sandusky Bay cHABs-Dominated by Planktothrix agardhii ...... 5

CHAPTER 2: MATERIALS AND METHODS ...... 7

2.1: Competition Experiments ...... 7

2.1.1: Culture Experimental Design...... 7

2.1.2: Monitoring the Growth of the Cultures ...... 9

2.1.2.1: Measurement of Growth Rates and Microcystis:

Planktothrix Ratio ...... 9

2.1.3: Determining Microcystin Concentrations ...... 9

2.2: Sediment Incubation Experiments ...... 10

2.2.1: Sample Site and Collection ...... 10

2.2.2: Nutrient Amendment Experimental Setup and Initial Nutrient

Analysis...... 11

2.2.3: Sampling for Enumeration/Chlorophyll Analysis ...... 12

2.2.4: Cell Enumeration ...... 13

2.2.5: Chlorophyll Extraction and Determining Chlorophyll Biomass ...... 13

CHAPTER 3: RESULTS ...... 15 vii

3.1: Competition Experiments ...... 15

3.1.1: Microcystis and Planktothrix Growth Rate ...... 15

3.1.2: The Influence of Nitrogen on the Ratio of Microcystis to Planktothrix 20

3.1.3: The Influence of Competition and Nitrogen Levels on Toxin

Accumulation ...... 22

3.2: Sediment Incubation Experiments ...... 23

3.2.1: Community Composition/Dominance ...... 23

3.2.2: Impacts of Nutrient Limitation ...... 27

CHAPTER 4: DISCUSSION ...... 30

4.1: Competition Experiments ...... 30

4.2: Sediment Incubation Experiments ...... 32

CHAPTER 5: FUTURE WORK ...... 34

LITERATURE CITED ...... 35 viii

LIST OF FIGURES

Figure Page

1 Schematic of trial setup for the reduced and nitrogen absent environments ...... 8

2 Map of Sandusky Bay ...... 11

3 Microcystis average growth rates when grown in monoculture and co-culture with

Planktothrix over three different nitrogen treatments based on cell enumeration ..... 15

4 Planktothrix average growth rates when grown in monoculture and co-culture with

Microcystis over three different nitrogen treatments based on cell enumeration ...... 16

5 Microcystis and Planktothrix growth when grown in monoculture over three

different nitrogen treatments based on cell enumeration ...... 18

6 Microcystis and Planktothrix growth when grown in monoculture over three

different nitrogen treatments based on in vivo chlorophyll fluorescence ...... 18

7 Microcystis to Planktothrix biovolume (µm3/mL) ratios in co-culture over three

different treatments ...... 20

8 Average rate of Microcystis to Planktothrix change per day over the course of two

trials per treatment ...... 21

9 Temperature of Sandusky Bay on each sampling day ...... 23

10 Dissolved nutrient concentrations in Sandusky Bay from early March thru late

April ...... 24

11 Total nutrient concentrations in Sandusky Bay from early March thru late April .... 24

12 Community composition in Sandusky Bay from late January thru late April ...... 25

13 Proportional breakdown of phytoplankton in the Sandusky Bay water samples...... 26

14 Proportional breakdown of cyanobacteria in the Sandusky Bay water samples ...... 27 ix

15 Cellular density of Planktothrix under four nutrient treatments at initial and final

(t=4) stages ...... 28

16 Amount of average chlorophyll biomass presented initially and at the end of

each nutrient amendment trial ...... 29

LIST OF TABLES

Table Page

1 Temperature of incubation for each trial of the sediment incubation experiment ..... 12

2 Probability value of significance of Microcystis monoculture vs co-culture from

each treatment ...... 15

3 Probability value of significance of monoculture vs co-culture from each treatment 17

4 Probability value of significance of Microcystis vs. Planktothrix monocultures

from each trial ...... 19

5 Average toxin accumulation of Microcystis, Planktothrix and combined cultures

at the different nitrogen treatments ...... 22

CHAPTER 1: INTRODUCTION 1.1: Shallow Nearshore Cyanobacterial Harmful Algal Bloom Environments

Lake Erie is one of the Laurentian Great Lakes that consist of five interconnected freshwater lakes holding ~20% of the world’s and 95% of the United States available surface freshwater supply. They are also one of the world’s largest and most biologically diverse freshwater resources (Magnuson et al., 1997). Lake Erie is the second smallest Great Lake by area and the smallest by volume. It is also the shallowest with an average depth of 20 m with the western basin of the lake only having an average depth of 7.4 m (Mortimer, 1987). It is surrounded by urbanized environment, vast expansive agricultural land use, and is therefore subject to anthropogenic stress and excessive nonpoint nutrient loadings (Fields, 2005). This causes cyanobacterial harmful algal blooms (cHABs) where different species of algae and cyanobacteria overgrow to the point where surface scums and benthic mats are formed. They can lead to hypoxia, reduced irradiance in the water column, and increased toxicity in the water.

(McKindles et al., 2020) The largest input of these nutrients has been documented from the

Maumee River on the southwestern edge of Lake Erie (Baker et al., 2014) with adjacent

Maumee Bay recording the highest concentrations of Chlorophyll-a (Chl-a) (Dolan et al., 2012,

Michalak et al., 2013, Steffen et al., 2014). Phosphorus loading was always believed to be a significant problem in all freshwater systems (Schindler et al., 1977) including western Lake

Erie, culminating in 1972 with the Great Lakes Water Quality Agreement (GLWQA), limiting the amount of total phosphorus that could be discharged in the lakes (Dolan, 1993). The

GLWQA helped reduce the Aphanizomenon-dominated cHABs that plagued the lake in the

1970s (McKindles et al., 2020, Steffen et al., 2014). However, beginning in the mid-1990’s, cHABs returned, marked by the identification of toxic Microcystis aeruginosa (Brittain et al.,

2000). Since then, these blooms have continued to be present and increase in severity (Davis et 2 al., 2019), including record blooms in 2011 and 2015 in addition to the “Toledo Water Crisis” event in 2014, which shut down the city’s public water system due to microcystins (cyclic hepatotoxins produced by Microcystis and Planktothrix) detected in finished drinking water

(Bullerjahn et al., 2016).

Sandusky Bay, an embayment of Lake Erie’s southern coast, is a drowned river mouth fed predominantly by the Sandusky River from the west and empties into Lake Erie to the east

(Salk et al., 2018). Covering approximately 65 km2 and with an average depth of 2m, it is a very hypereutrophic system receiving large amounts of nutrient input from the surrounding agricultural landscape (Salk et al., 2018). The shallow waters of Sandusky Bay lead to very effective wind-driven mixing, creating very turbid water (Scheffer et al., 1997). This is also explained by the sediment delivered from the river being very loose, which contributes to poor light penetration and high particulate nutrient loading (Davis et al., 2015). The bay consists of many species of diazotrophic (nitrogen fixing) cyanobacteria, such as Dolichospermum,

Aphanizomenon, Cuspidothrix and Cylindrospermopsis, but is dominated by Planktothrix agardhii, a non-diazotrophic species (Conroy et al., 2005, Davis et al., 2015, Salk et al., 2018).

1.2: Composition of Western Lake Erie cHABs

Microcystis is a cyanobacterium that is globally relevant, having been reported blooming on every continent, except Antarctica (Zurawell et al., 2005). Many of its strains are known to produce toxins, mostly the cyclic hepatotoxins microcystins, but a few have been reported to produce neurotoxins, such as anatoxin-a, originally known as fast-death factor (Bishop et al.,

1959, Park et al., 1993). This genus is characterized morphologically by cells that are highly buoyant, unicellular, colony-forming and coccoid-shaped, with a diameter ranging from 1 to 9

µm (Komarek et al., 2002). Microcystis also can regulate its buoyancy in many eutrophic, highly 3 turbid, and potentially light limited environments, allowing it to be a bloom-dominating organism. They can do this through formation and collapse of intracellular gas vesicles (Walsby et al., 1994). They can sink to access nutrient rich bottom waters and float to optimize utilization of radiant energy (Hark et al., 2016).

Planktothrix agardhii is a species of cyanobacterium that is not as globally distributed as

Microcystis, mainly being found in the northern hemisphere (Suda et al., 2002). It prefers shallow, well-mixed, nutrient-rich lakes normally found in temperate climate zones (Kokocinski et al., 2011). Many of its strains produce microcystins, specifically the desmethyl microcystins

(Lindholm et al., 2011, Luukkainen et al., 1993) These are filamentous cyanobacteria characterized by solitary trichomes that are straight or slightly curved with cylindrical cells that are slightly shorter than wide. Filaments can contain hundreds to thousands of cells and are less than 6 µm in diameter with varying lengths that extend up to a few mm (Kurmayer et al., 2016).

Like Microcystis, they can also regulate their buoyancy, doing this via a gas-filled space within the protoplast. This gas vesicle form has a density approximately one-tenth of water (Walsby et al., 1994).

1.3: Physicochemical Factors That Affect cHABs

Factors, such as temperature, nutrients, and light are crucial to the forming of cHABs.

Both species can persist in highly turbid environments, such as Sandusky Bay and, to a lesser extent, Western Lake Erie. Microcystis generally prefers environments with higher nutrient, lower turbulence, and higher temperatures than Planktothrix and generally has a higher light tolerance (Nõges et al., 2003, Post et al., 1985). However, Microcystis can also be an excellent scavenger for phosphorus which can explain why these blooms can be found at lower dissolved phosphorus concentrations. (Harke et al., 2016) Planktothrix can grow over a much broader 4 temperature range than Microcystis. Microcystis is known to thrive between 24-34°C, however

Planktothrix can survive and grow in far lower temperatures, even below 10°C (Imai et al., 2008,

Nõges et al., 2003, Post et al., 1985).

Allelopathic chemicals are also produced by both Microcystis and Planktothrix as a method of inference competition. (Gross et al., 2012) These allelochemical reactions are described for phytoplankton that include cell lysis, reactive oxygen species production, and inhibition of photosynthesis, enzyme activity, and nucleic acid synthesis. (Legrand et al., 2003,

Leflaive et al., 2007). Microcystis produces less of these chemicals in monoculture than

Planktothrix but more in co-culture. These chemicals negatively effect Planktothrix including decreasing in trichome size or alterations in morphology of cells (Briand et al., 2019).

Two crucial limiting nutrients that greatly affect the formation and the maintenance of these blooms are nitrogen species (nitrate, ammonia and urea) and phosphate. The Redfield

Ratio, which is based on the elemental composition of phytoplankton reveals the optimal stoichiometry of carbon, nitrogen, and phosphorus for growth to be 106 mol C: 16 mol N: 1 mol

P.(Redfield, 1993, Geider et al., 2002) While many lakes have traditionally been viewed to be

P-limited (Schindler et al., 1977), Sandusky Bay is often N limited during much of the summer and early fall due to high rates of denitrification (Davis et al., 2015, Salk et al., 2018). Yet the blooms continue to proliferate due to these non-diazotrophic species abilities to access and store nutrients while maintaining high photosynthetic rates (Hampel et al., 2019).

1.4: How Planktothrix Acclimates to Low Nitrogen Environments

Even in low nitrogen environments, such as Sandusky Bay, Planktothrix agardhii dominates throughout the bloom season. In Lake Taihu, Microcystis can outcompete diazotrophs 5 or cyanobacteria that fix atmospheric nitrogen into more useable forms as nitrogen fixation is an energy demanding process that requires access to consistent light levels to achieve high rates of photosynthesis (Blomqvist et al., 1994, Kappers et al., 1980). Recent studies on the nitrogen depleted waters of Sandusky have found Planktothrix to be a more efficient scavenger of nitrogen than Microcystis (McKindles et al., 2020). Planktothrix like most cyanobacteria store nitrogen in a molecule known as cyanophycin, a polypeptide consisting of aspartic acid as a backbone and arginine as a side chain (Hampel et al., 2019, Tseng et al., 2013). Under conditions of luxury consumption of nitrate, it is manufactured via the enzyme cyanophycin synthetase (Hai et al., 1999, Hampel et al., 2019). This enzyme mobilizes stored nitrogen when nitrogen sources in the water column are depleted (Hampel et al., 2019). Planktothrix is also an efficient scavenger of ammonium during periods of N-depletion, yielding a Km for ammonium

2-5-fold lower than Microcystis (Hampel et al., 2019). These data suggest that Planktothrix is better adapted to environments prone to N-limitation.

1.5: Sandusky Bay cHABs-Dominated by Planktothrix agardhii

Between the months of May to October, Planktothrix is the dominant phytoplankton, with the rest of the cyanobacterial biomass (up to 20%) contributed by nitrogen-fixing species

(28). This is likely due to N:P ratios in Bay waters being far lower than offshore sites in Western

Lake Erie which many times can trend below the 16:1 Redfield Ratio in the Bay during the summer (Davis et al., 2015). There are two main goals of this thesis studying Planktothrix in

Sandusky Bay. The first will focus on how Planktothrix and Microcystis may compete in so- called mixing zones, such as where Sandusky Bay meets Western Lake Erie. Given the results of prior studies, I hypothesize that Planktothrix will be dominant over Microcystis when nitrogen is the limiting nutrient and that Microcystis will be dominant in nitrogen replete conditions. 6

Knowledge of bloom activity during the summer months in Sandusky Bay is well documented, however less knowledge exists in the winter and the spring leading up to these expansive blooms. The second goal focuses on the bay during winter and spring, where it is hypothesized that Planktothrix is the dominant phytoplankton in Sandusky Bay which most likely expresses N limitation during these seasons. 7

CHAPTER 2: MATERIALS AND METHODS

2.1: Competition Experiments

2.1.1: Culture Experimental Design

The three types of environments simulated by these experiments were ones with replete nitrogen, reduced nitrogen (about 10% of the nitrogen present in the replete environment), and fully depleted nitrogen. Jaworski’s Medium (JM) (Culture College of Algae and Protozoa, see https://www.ccap.ac.uk/pdfrecipes.htm) was used to represent the replete environment whereas modified JM (0.111mM and 0 M NO3) was used to represent the latter two.

Cultures of Microcystis aeruginosa LE-3 (Brittain et al., 2000) and Planktothrix agardhii

1033 (culture started by Emily Davenport in the lab of Dr. George S. Bullerjahn in 2016, characterized by Rainer Kurmayer at the University of Innsbruck, Austria) were grown at 25°C in JM in stock cultures before sub-culturing. The goal for the mixed cultures was cellular inoculation of Microcystis to Planktothrix (M:P) ratios between 1:2 and 1:1. Trials consisted of

18 sterile 35 mL polycarbonate tubes split into three groups of six tubes: six containing only

Microcystis, six containing only Planktothrix, and six containing a combination of Microcystis and Planktothrix. Cultures that were being inoculated into the JM environment had Microcystis and Planktothrix added into monoculture tubes. After one week of growth, the co-culture tubes were made by obtaining six fresh tubes and adding 15 mL of each monoculture into the new combined tubes. Cultures that were going to be introduced into the modified JM environments 8 were collected by centrifugation at 5000 g for 5 min and washed with their respective media three times. The trials were then set up according to the schematic detailed in Figure 1.

Figure 1: Schematic of trial setup for the reduced and nitrogen absent environments

(*Concentration of Nitrogen in JM is either 0.111mM or 0M)

The trials were then placed in a 25°C incubator that had a 12-hour day: night light cycle with light levels at 100 µmol m-2 s-1. All trials ran for approximately 1.5 weeks, except for the second trial of the nitrogen depleted treatment (see Figure 7d) where an additional goal was to see which cyanobacterial genus started to decline due to nitrogen limitation 9

2.1.2: Monitoring the Growth of the Cultures

Cell growth was monitored during the experiment by measuring raw chlorophyll fluorescence as a proxy for biomass on a Turner Designs TD-700 Fluorometer at intervals that ranged from daily to every 3 days on the longer running trial. The 35 mL polycarbonate tubes are accommodated by the fluorometer sample chamber, allowing measurements to be taken efficiently and non-invasively. Once measured for fluorescence, 1 mL was withdrawn from each tube (daily for co-cultures, every 3 days for monocultures) and fixed with 20 µL of Lugols iodine until cell enumeration could be performed. Cellular enumeration was performed using a

Neubauer hemocytometer with cells being counted on an inverted microscope.

2.1.2.1: Measurement of Growth Rates and Microcystis: Planktothrix Ratio

Growth Rates (day-1) for Microcystis and Planktothrix were calculated when in monoculture and co-culture using a linear regression on the natural log of the exponential growth phase of each trial. For the co-cultures with Microcystis and Planktothrix, cellular biovolume

(µm3 mL-1) was calculated for each and then plotted as a ratio of Microcystis to Planktothrix biovolume for each treatment. The change in this Microcystis to Planktothrix ratio over time was calculated using a linear regression for each trial and then averaging the slopes from these regressions together to determine if the M:P ratio was increasing or decreasing over time.

2.1.3: Determining Microcystin Concentrations

Samples to be measured for microcystins were taken at timepoints at the beginning, middle, and end of the experiment, except for the longer running trial where it was taken at the beginning, then twice a week until the conclusion of the experiment. This was done by taking

100 µL of biomass from each of the six replicates into a glass vial so in the end there were three 10 pooled toxin vials each with 600 µL representing the three variables tested. Before analysis, the vials underwent three freeze-thaw cycles to lyse cells to ensure complete toxin extraction as per

EPA method 546. The levels of microcystins were determined using the Abraxis-Microcystins-

ADDA ELISA method as per manufacturer instructions.

2.2: Sediment Incubation Experiments

2.2.1: Sample Site and Collection

All samples were collected from a dock at Clemons Boats Marina in Sandusky, OH

(41.469167°N, -82.818611°W). This sample site was chosen because it is approximately at the location where inner and outer Sandusky Bay meet, therefore minimizing the influence from the

Sandusky River inlet to the west and the Lake Erie outlet to the east (Figure 1). On each sampling occasion, water was collected using three 20 L acid-washed carboys. Sediment was collected with a Ponar Grab Sampler and the entire grab stored in acid-washed 1-L bottles on ice for transport to the lab. 11

Figure 2: Map of Sandusky Bay (The red dot indicates the location of Clemons Marina.)

(Credit: Google Maps)

2.2.2: Nutrient Amendment Experimental Setup and Initial Nutrient Analysis

Equal volumes from each of the three carboys were combined into a single 20 L pooled sample. The water was then stored in a 4°C dark refrigerator for 24h to allow material in the water column to settle. Experimental setup included 12 x 250 mL polycarbonate PE bottles that had 200 mL of lake water decanted from the top of the pooled sample and 50 mL of sediment.

Sediment bottles were divided into four treatments in triplicate including a control (no nutrients added) and three treatments spiked with the following nutrients: nitrate (50 µM NaNO3), phosphate (2 µM Na2HPO4*7H2O), and a combined treatment containing both N+P(50 µM

NaNO3, 2 µM Na2HPO4*7H2O). Sample bottles were then incubated in a 12 hour light: dark cycle with a light intensity of 100 µmol m-2sec-1 at different temperatures as indicated in Table 1 to assess the response of the phototrophic communities as the temperature was increased, mimicking the transition from winter to spring in Sandusky Bay. 12

Lake water from the combined pooled sample was also collected for nutrient analysis.

For dissolved nutrient analysis, this water was filtered through 0.2 µm Sterivex filters before being collected. Both waters collected for dissolved and total nutrient assays were frozen until being analyzed on a SEAL multichannel autoanalyzer in the lab by Dr. Justin Chaffin, Research

Coordinator and Senior Researcher at The Ohio State University Franz Theodore Stone

Laboratory.

Table 1: Temperature of incubation for each trial of the sediment incubation experiment

Time of 1/25-2/15 2/6-3/1 2/19-3/14 3/6-3/29 4/2-4/26 4/16-5/9 4/30-5/24

Incubation

Incubation 4 10 15 15 22 22 22

Temperature

(°C)

2.2.3: Sampling for Enumeration/Chlorophyll Analysis

Throughout the four weeks that each trial was run, bottles were sampled twice a week, totaling eight sampling periods. The initial samples were collected from three random bottles out of the twelve after the whole water was added but before the sediment or nutrients were added.

Every other sampling was done from all twelve bottles.

For cell enumeration, 10 mL of whole water was placed in a 15 mL Falcon tube with 200

µL of Lugols Iodine (1.3% final concentration). The water was withdrawn using a 10 mL pipette and care was taken to move the pipette through the water column to ensure a representative sample was being taken without having to shake the sample and disturbing the sediment. For chl- a, 10 mL of whole water was extracted using the same procedure as described above and cells 13 concentrated using 0.2 µm pore size, 25 mm polycarbonate filters under gentle vacuum. Filters were stored in 15 mL Falcon tubes and frozen until analysis.

2.2.4: Cell Enumeration

This component of the analysis was completed by William Cody of Aquatic Taxonomy

Specialists. Due to resource and time constraints, only the initial and final samplings were enumerated by microscopy, with the triplicate initial samples being combined/concentrated before enumeration. In addition, only one of the triplicates of each treatment of the final samples was enumerated. This explains the lack of error bars on figures 12-14. All aliquots for analysis were 1 mL and analyzed at 400x along two complete scans or strips across the bottom of the inverted microscope chamber. All species of algae were enumerated, except for nanoplankton as they are difficult to accurately see and enumerate at 400x. Trichomes of Planktothrix of each strip were separated into categories or groups of short and long individuals. Short trichomes contained 10 to 50 cells and long trichomes 50+ cells. Density of short and long trichomes per milliliter were calculated and these numbers were multiplied by the respective mean number of cells in each category or group of trichome length. Mean number of cells for short and long trichomes were calculated by randomly counting ten trichomes in each category and taking the median number of cells. The total cell density of the short and long categories was added together for total cell density.

2.2.5: Chlorophyll Extraction and Determining Chlorophyll Biomass

Chl-a was extracted with 90% aqueous acetone solution. The tubes were then placed in a

-20°C freezer for 24h for extraction. Following clarification by centrifugation at 3000 x g for 5 minutes, the supernatant was transferred into a glass culture tube and the initial chl concentration 14

(µg L-1) was read on a Turner Designs TD-700 fluorimeter. Samples yielding readings above the instrument calibration limit were then diluted with the acetone solution and reread. The final chl

푣 퐶ℎ푙 = ( 𝑖 [ ]) 퐷 concentrations were calculated using the following formula: 푎 where i was the initial chl concentration, v was the filtered volume (mL), a was the acetone volume added (mL) and D was the dilution factor. 15

CHAPTER 3: RESULTS

3.1: Competition Experiments

3.1.1: Microcystis and Planktothrix Growth Rate

0.5

0.4 ) 1 - 0.3

0.2

0.1 Growth Rate (d Rate Growth 0 JM JM10%N JM0%N -0.1 Trial

Co-Culture With Planktothrix Microcystis Monoculture

Figure 3: Microcystis average growth rates when grown in monoculture and co-culture with

Planktothrix over three different nitrogen treatments based on cell enumeration

Table 2: Probability value of significance of Microcystis monoculture vs co-culture from each treatment Trial P Value (One Way ANOVA)

JM 0.28

JM 10% 0.16

JM 0% 0.367 16

There seems to be no statistical differences in growth rate when Microcystis is growing in monoculture instead of in co-culture with Planktothrix. (Figure 3 and Table 2). The restriction of nitrogen does not seem to affect Microcystis growth rates either. Only when nitrogen is eliminated from the environment does Microcystis growth rate both in co-culture and in monoculture significantly decrease. (Figure 3)

0.25

) 0.2 1 -

0.15

0.1

Growth Rate (d Rate Growth 0.05

0 JM JM10%N JM0%N Trial

Co-Culture with Microcystis Planktothrix Monoculture

Figure 4: Planktothrix average growth rates when grown in monoculture and co-culture with

Microcystis over three different nitrogen treatments based on cell enumeration 17

Table 3: Probability value of significance of monoculture vs co-culture from each treatment

(Bold P values indicate statistical significance)

Trial P Value (Single Factor Anova)

JM 0.29

JM 10% 0.04

JM 0% 0.07

Like Microcystis, Planktothrix does not seem to grow any differently if it is in monoculture versus co-culture with Microcystis under nitrogen replete conditions. However, at restricted nitrogen levels, it seems to grow much better alone than when it is competing for the nutrient with Microcystis (Figure 4 and Table 3). When no nitrogen is present, however the growth difference between monoculture and co-culture seems to be absent. 18

0.5

0.4 ) 1 - 0.3

0.2

0.1 Growth Rtae Rtae (d Growth

0 JM JM10%N JM0%N -0.1 Trial

Microcystis Monoculture Planktothrix Monoculture

Figure 5: Microcystis and Planktothrix growth when grown in monoculture over three different nitrogen treatments based on cell enumeration

0.35 0.3 1) - 0.25 0.2 0.15 0.1 Growth Rate (d Rate Growth 0.05 0 JM JM10%N JM0%N Trial

Microcystis Monoculture Planktothrix Monoculture

Figure 6: Microcystis and Planktothrix growth when grown in monoculture over three different nitrogen treatments based on in vivo chlorophyll fluorescence 19

Table 4: Probability value of significance of Microcystis vs. Planktothrix monocultures from each trial (Bold P values indicate statistical significance)

Trial P Value (Single Factor Anova) P Value (Single Factor Anova) Figure 5 Figure 6

JM 0.46 0.91

JM 10% 0.67 0.14

JM 0% 0.50 0.01

Comparing growth rates of Microcystis and Planktothrix in monocultures using cellular enumeration lead to no significant differences in growth at any of the treatments. The only significant difference is that growth of both species decreased when nitrogen was removed from the environment (Figure 5). However, when their growth rates were compared using in vivo chlorophyll florescence data there was a significant difference that Planktothrix grew more efficiently in a nitrogen absent environment than Microcystis (Figure 6 and Table 4). 20

3.1.2: The Influence of Nitrogen on the Ratio of Microcystis to Planktothrix

Figure 7: Microcystis to Planktothrix biovolume (µm3/mL) ratios in co-culture over three different treatments. Treatments include a) Replete Nitrogen b) Restricted Nitrogen and c-d) No Nitrogen*

*Trial 2 is graphed separate of Trial 1 of no nitrogen due to being performed over a much longer span of time

When Microcystis and Planktothrix are in co-culture, in most treatments, the ratio of

Microcystis to Planktothrix increased, indicating that Microcystis can outcompete Planktothrix over time (Figure 4) in both trials with replete N. 21

0.07 0.06 0.05 0.04 0.03 0.02

M:P Ratio Rate/day M:P Ratio 0.01 0 JM JM 10%N JM 0%N -0.01 Trial

Figure 8: Average rate of Microcystis to Planktothrix change per day over the course of two trials per treatment

However, as nitrogen was reduced, the rate of the Microcystis to Planktothrix ratio over time also decreased (p = 0.005) (Figure 8). 22

Table 5: Average toxin accumulation of Microcystis, Planktothrix and combined cultures at the different nitrogen treatments

JM (ppb) JM 10%N (ppb) JM 0%N (ppb)

Microcystis 2.46 (SD = 1.30) 77.94 (SD = 60.34) 1.92 (SD = 0.95)

Planktothrix 3.93 (SD = 2.42) 53.44 (SD = 96.14) 15.71 (SD = 5.30)

Combined >1000* (n=6) 131.25 (SD = 4.19 (SD = 5.98)

131.77)

3.1.3: The Influence of Competition and Nitrogen Levels on Toxin Accumulation

Measuring total microcystins equivalents by the ELISA method, toxin concentration at the conclusion of each trial revealed that in nitrogen replete conditions, the co-culture trials yielded the highest concentration of microcystins. The effect disappears as nitrogen is reduced

(Table 5). In the nitrogen-free treatment, Planktothrix in monoculture produces significantly more toxin than Microcystis in monoculture or in co-culture. 23

3.2: Sediment Incubation Experiments

3.2.1: Community Composition/Dominance

14 12 º) 10 8 6 4 Temperature (C Temperature 2 0 25-Jan 6-Feb 19-Mar 4-Mar 1-Apr 16-Apr 29-Apr Date

Figure 9: Temperature of Sandusky Bay on each sampling day. Arrow indicates first sampling date after ice-out.

The water temperature of Sandusky Bay at the Clemons Marina stayed consistent throughout the winter season, and only after ice-out the bay’s water temperature increased to

12°C (Figure 9). 24

200 180 160 140 120 100 80 60 Concentration (µg/L) Concentration 40 20 0 2/24/2019 3/6/2019 3/16/2019 3/26/2019 4/5/2019 4/15/2019 4/25/2019 5/5/2019 Date Sampled

Nitrate +NO2 Ammonium Nitrite DRP Silicate Nitrate

Figure 10: Dissolved nutrient concentrations in Sandusky Bay from early March thru late April.

300 250 200 150 100 50 Concentration (µg/L) Concentration 0 2/24/2019 3/6/2019 3/16/20193/26/2019 4/5/2019 4/15/20194/25/2019 5/5/2019 Date Sampled

Total Phosphorus Total Kjeldahl Nitrogen Total Nitrogen Total Nitrogen:Total Phosphorus

Figure 11: Total nutrient concentrations in Sandusky Bay from early March thru late April. 25

Nutrient analysis of Sandusky Bay showed overall that the bay was Phosphate limited for much of the spring (Figures 10 and 11). Dissolved nutrient analysis revealed that the primary forms of nitrogen present were nitrite and nitrate while more reduced forms, such as ammonium were limited. In addition, there were high amounts of dissolved silicate present in the water that declined sharply through the month of April (Figure 10).

100000

10000

1000

Cells/mL 100

1 1/25/2019 2/6/2019 2/19/2019 3/6/2019 4/2/2019 4/16/2019 4/30/2019. Date Sampled

Green Algae Diatoms Cyanobacteria Cryptomonads Other

Figure 12: Community composition in Sandusky Bay from late January thru late April (Plotted on a logarithmic scale)

The initial winter samples yielded low phytoplankton cell densities until the April sampling dates, when cell abundances exceeded 1.6 x 105. The March and Early April dates revealed an early spring diatom bloom preceding dominance by cyanobacteria in late April

(Figures 12 and 13). 26

1 0.9 0.8 0.7 0.6 0.5 0.4 0.3

Percent Abundance Percent 0.2 0.1 0 1/25/2019 2/6/2019 2/19/2019 3/6/2019 4/2/2019 4/16/2019 4/30/2019. Date Sampled

Green Algae Diatoms Cyanobacteria Cryptomonads Other

Figure 13: Proportional breakdown of phytoplankton in the Sandusky Bay water samples

In addition to the early spring diatom bloom and dominance by cyanobacteria in late spring, cyanobacteria were the dominant phytoplankton among the community that was present in winter (January), followed by cryptomonads in a February sample that preceded the spring diatom bloom (Figure 13). 27

1 0.9 0.8 0.7 0.6 0.5 0.4 0.3 Percent Abundance Percent 0.2 0.1 0 1/25/2019 2/6/2019 2/19/2019 3/6/2019 4/2/2019 4/16/2019 4/30/2019. Date Sampled

Chroococcus microscopicus Merismopedia Microcystis Planktolyngbya Plantothrix agardhii

Figure 14: Proportional breakdown of cyanobacteria in the Sandusky Bay water samples

Knowing that cyanobacteria dominated in the middle of the winter, late spring and into the summer (Conroy et al., 2005, Salk et al., 2018), it is clear that Planktothrix agardhii, regardless of season or temperature is the dominant cyanobacterial species in Sandusky Bay

(Figure 14). The third trial from 2/19-3/14 had no cyanobacteria enumerated, which explains the absence of the data.

3.2.2: Impacts of Nutrient Limitation

The addition of spiking the bottles with either phosphorus, nitrate, or both was to explore our hypothesis that Sandusky Bay exhibits N limitation. This was analyzed using not only the 28 initial samples but samples that were incubated for four weeks under the various nutrient conditions.

1.00E+07 1.00E+06 1.00E+05 1.00E+04 1.00E+03 Cells/mL 1.00E+02 1.00E+01 1.00E+00 1/25-2/5 2/6-3/1 2/19-3/14 3/6-3/29 4/2-4/26 4/16-5/9 4/30-5/24 Trial Duration

Initial Control Nitrate Phosphate Nitrate and Phosphate

Figure 15: Cellular density of Planktothrix under four nutrient treatments at initial and final (t =

4 weeks) stages. (Plotted on a logarithmic scale)

The first trials where the temperatures of incubation were between 4 and 10°C (See Table

1) there was no nutrient limitation observed, as cell counts indicated that the unamended controls grew as well as all other treatments (Figure 15). In the late winter (February and early March) trials, the treatments reveal higher growth when nitrogen was added to the samples. During the latter half of the experiment when the temperature of incubation was increased to 22°C, the samples appeared to be co-limited by both nitrate and phosphate (Figure 15). 29

140 120 100 80 60 40 Chlorophyll (µg/L) Chlorophyll 20 0 1/25-2/15 2/6-3/1 2/19-3/14 3/6-3/29 4/2-4/26 4/16-5/9 4/30-5/24 Trial Duration

Initial Control Nitrate Phosphate Nitrate and Phosphate

Figure 16: Amount of average chlorophyll biomass presented initially and at the end of each nutrient amendment trial

Using chlorophyll as a proxy for total biomass revealed that the later trials incubated at a warmer temperature (22°C) suggest possible nitrate limitation (4/2-4/26, p = 0.03) but not colimitation with phosphate. All other trials preceding it seem to have no other nutrient limitation. The most important variable promoting growth was light, as the unamended controls in all trials yielded an increase in chlorophyll above the initial measurement at the start of the incubation (Figure 16). 30

CHAPTER 4: DISCUSSION

4.1: Competition Experiments

The growth rates in monocultures of Microcystis and Planktothrix followed different patterns compared to mixed cultures. Nitrogen appeared to have no effect on Microcystis growth in monoculture except in the nitrogen-free medium, in which its growth rate was substantially reduced. This suggests that Microcystis can efficiently scavenge nitrogen when it is scarce. It didn’t appear that growing in monoculture vs co-culture had any effect on Microcystis growth rate. Nitrogen didn’t have an effect in monoculture for the growth rate of Planktothrix until the nitrogen was removed from the environment, like Microcystis. However, at reduced nitrogen,

Planktothrix had a much lower growth rate when grown with Microcystis than growth alone.

This could result from a production of intracellular compounds and allelochemicals by

Microcystis which it is known to produce more under co-culture conditions with Planktothrix than in monoculture (Briand et al., 2019). In addition, this could also be the result from less nitrogen being present than in the replete environment, however the concentration of nitrogen in this environment is 110 µM, which is in the range of concentrations of nitrogen in Sandusky Bay during the spring, averaging in the range of 200 µm (Conroy et al., 2015, Salk et al., 2018).

However, these differences in growth rate did not translate to the nitrogen replete or depleted environments. This is likely due to the lack of nutrient limitation in the replete environment or the extreme stress in no nitrogen environment where the presence or absence of Microcystis carries less significance. Comparing Microcystis and Planktothrix growth in monocultures, the only significant difference was found when Planktothrix grew better in the no nitrogen environment, most likely due to the ability of Planktothrix’s to thrive in nitrogen poor environments. 31

When examining the competing cultures, it is clear that in all three treatments over time that Microcystis was dominant over Planktothrix as the M:P ratio rose in all three cases. As nitrogen was reduced in each treatment, that ratio also declined indicating that Microcystis was not as dominant over Planktothrix. It is known that Planktothrix does in fact scavenge nitrogen better than Microcystis (Hai et al., 1999, Salk et al., 2018). In sum Planktothrix persists in a low nitrogen environment but it does not appear that competition for nitrogen drives Planktothrix success over Microcystis in these experiments. Future experiments should be focused on not only nutrients, but the interplay of light and nutrients, knowing that Planktothrix is well-adapted to low light environments (Post et al., 1985).

The experiments also suggest that for both Microcystis and Planktothrix, stressors led to the increased production of microcystins. In N replete JM medium that stressor is competition as there was significantly more toxin produced when the two genera were combined compared to when they were grown separately. Under conditions of depleted nitrogen, Planktothrix in monoculture produced more toxin than the Microcystis monoculture or the combined culture (p =

3.87 x 10-5). In addition these experiments were all done at an irradiance of 100 µmol m-2sec-1 which is a very strong light intensity and at a single temperature of 25°C. Planktothrix is known to tolerate a broader range of temperatures and lower light intensities, therefore in the future these experiments should be done over a broader range of temperature and light intensities to determine under which conditions each genus outcompetes the other. It’s likely that light or temperature influences the community composition since it is not nitrogen at the light intensities and temperatures employed in this study. 32

4.2: Sediment Incubation Experiments

The temperature of Sandusky Bay throughout the sampling period was static around 2°C until around the spring when for the last three trials in April had the temperature steadily increased by approximately 10°C over the month. The steep rise in water temperature closely following increased air temperature can be attributed to the very shallow depth of the sampling site in Sandusky Bay. The rise in temperature also correlates with increasing cell density of the microalgal communities. It also revealed an increase in the diatom population throughout the month of April before the diatoms essentially disappeared leaving just cyanobacteria, explaining the sharp drop in cell density in the last trial. The rise and fall of diatom populations in April also correlates with the decline of dissolved silica measured in Lake Erie during that month. It was also determined that Planktothrix was the dominant cyanobacterium as has been shown to be the case in the past (Salk et al., 2018). When examining the community breakdown overall, even though cellular density was very low in January, Planktothrix dominated in the winter and spring. A transitional period of cryptomonad abundance preceded the early spring diatom bloom that in turn preceded the spring-summer dominance of Planktothrix. This cryptomonad is consistent with a previous study done in 2015 showing cryptomonads and green algae being the most abundant in the water sampled (Collier et al., 2016)

Over this period there were two types of nutrient limitation observed. Analyzing the response of Planktothrix to the nutrient amendment experiment revealed a slight nitrate limitation during the fourth trial before the cellular biomass increased in the beginning of April where it seems that there was both a nitrate and phosphate limitation. Expanding to chlorophyll biomass which considers all the phytoplankton, there was no nutrient limitation before the cell abundance increased in the beginning of April. At that point it appeared to be nitrate limited, but 33 not phosphate limited as seen in the Planktothrix enumeration figures. This contradicts both dissolved and total nutrient data analyzed that revealed that phosphorus was the limiting nutrient.

The nutrient amendment experiment did not have any statistical significance due to no replicates being analyzed. Further analysis of these replicates could align with the nutrient data. The nutrient data would explain a previous study completed in Sandusky Bay in 2015 showing that phosphorus alone stimulated growth to levels higher than unamended controls (Collier et al,

2015). Overall the data implies that the communities in Sandusky Bay are overall nitrate limited but nitrogen is not the main driver that influences cyanobacterial community composition under the light and temperature conditions assessed in this thesis. 34

CHAPTER 5: FUTURE WORK

It is clear from the results that under the experimental conditions, Microcystis has more rapid uptake kinetics of nitrogen than Planktothrix, although that uptake rate declined as nitrogen became increasingly scarce in the environment. In the future, different light levels should be tested, especially since Planktothrix is a low light-adapted cyanobacterium. Trials at different temperatures can also be run to see how that affects the Planktothrix and Microcystis relationship as Planktothrix has a greater temperature tolerance range than Microcystis. When preparing the experiments, nitrogen starvation of the cyanobacteria prior to inoculation into the different nitrogen concentrations could also be as they have been in previous experiments (Tan et al.,

2019). Testing other strains of both Microcystis and Planktothrix should be performed to tell us if the results are consistent across various toxigenic and nontoxigenic strains. Finally, further analyzing microcystins congeners from toxin samples taken from the combined incubations should be completed to evaluate how much toxin Microcystis and Planktothrix (methylated vs. desmethylated microcystins) each produced. This can reveal whether one cyanobacterium is responsible for the increase in toxin in mixed culture. 35

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