The Effect of Temperature on the Toxicity of Planktothrix Agardhii
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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 © 2019 Christina Moore All Rights Reserved iii 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 1 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.