Sustainable Methods for Cyanotoxin Treatment and Discovery of the Cyanophage THESIS Presented in Partial Fulfillment of the Requirements for the Degree Master of Science in the Graduate School of The Ohio State University By Xuewen Jiang Graduate Program in Food Science and Technology The Ohio State University 2017 Master's Examination Committee: Jiyoung Lee, Advisor Ahmed Yousef Luis Rodriguez-sonata Copyrighted by Xuewen Jiang 2017 Abstract Cyanobacterial harmful algal bloom (CHABs) has been a great concern due to the detrimental effects on ecosystem, animal and human health. Overgrowth of CHABs causes hypoxia and some species produce unpleasant odor compounds. More importantly, multiple toxins (cyanotoxins) produced by CHABs can be harmful to animal and human health by damaging internal organs, including liver, kidney, reproductive and neurological systems. Water-mediated activities are believed to be the common exposure routes for human to these toxins. Many studies reported that these toxins can be accumulated into food or dietary supplements via contaminated water, since the guideline for agriculture and aquaculture water use is not established yet. Therefore, strategies for controlling both CHABs and their toxins are important and imperative for protecting environmental and food safety. Chapter 1 summarized the literature review about CHABs and their toxins, and identified knowledge gaps. Furthermore, previous research regarding to cyanophages and their ecological roles are also reviewed because understanding of cyanophage, natural predators of cyanobacteria, is critical to understanding the bloom formation and evolution. Among the cyanotoxins, the most abundant cyanotoxin is microcystin (MC), which is a seven-amino-acid peptide, consisting of more than 150 congeners. MC shows toxicity via inhibiting protein phosphatase 1 and 2A, and generate reactive oxygen species (ROS), leading to cell apoptosis in the involved organs. MCs are chemically stable against ii sunlight, extreme pH and boiling, therefore, it is difficult to treat the contaminated water once MCs are released. Physical, chemical and biological methods have been applied to treat MCs in water, but few of them can be successfully applied for MC- contaminated food. New technologies for MC treatment are proposed in Chapter 2. The objective of Chapter 2 was to develop sustainable and non-chemical-based methods for controlling MCs: 1) ultraviolet with TiO2, which is a common food additive as well as a photocatalyst; and 2) cold plasma, which is a non-thermal treatment using ionized gas. Natural MCs were extracted from Microcystis aeruginosa and treated with several combinations: 1) UV at intensity of 1470 μW/cm2 [high] or 180 μW/cm2 [low]; 2) cold plasma; and 3) no treatment in a dark room (control). To determine synergistic effects, nanoparticles (TiO2) were coated on the outside of the UV treatment chamber prior to irradiation. The MC degradation efficiency was enhanced by the reusable TiO2 coating at lower UV intensity by 10 percent, but no significant difference was observed at higher intensity of UV. Cold plasma removed MCs rapidly (80% and 92% in 1 and 2 hours, respectively) under experimental conditions, indicating that it can be easily and practically used in household and industrial setting. In environmental settings, controlling CHABs formation is a better and effective way to reduce toxin production. Many previous studies have focused on environmental factors on blooms, but the role of cyanophage (viruses whose hosts are cyanobacteria) in the ecology of toxin production is much less understood, especially in freshwater. Cyanophage are known to contribute to biological controlling of CHABs by influencing the metabolism and evolution of cyanobacteria. Therefore, the information on the iii characteristics of cyanophage infection is crucial in potential bloom control. In Chapter 3, findings about cyanophage isolated from Lake Erie during bloom season were reported. The objectives of this study are to 1) isolate and characterize cyanophages from Lake Erie; and 2) examine the host-cyanophage interactions using multiple tools, especially atomic force microscopy (AFM). Cyanophage isolates were inoculated into toxin- producing Microcystis aeruginosa (host) originated from Lake Erie. The dynamic interactions between phages and hosts were monitored using spectrophotometer, fluorimeter, quantitative PCR and AFM. The structural and genetic characters of the cyanophages were identified by using transmission electron microscope (TEM) and PCR. The Podoviridae ~300 nm in size were infectious against the toxic strain of M. aeruginosa. The growth and photosynthesis capacity of hosts was significantly inhibited after cyanophage infection and cellular damages on membranes over time were clearly observed. We also found that UV irradiation with proper dose can induce the cyanophage shifting from their lysogenic stage to lytic stage. The psbA (photosynthesis core protein A) and the gp58 (special capsid protein) genes were identified, which showed high similarities of the same genes identified in marine cyanophages, indicating a close evolutionary root. In summary, this thesis reports comprehensive knowledge about CHABs and their toxins. In addition, emerging techniques were applied in this study to provide informative and essential basis to develop controlling strategies in various settings when toxins are present. iv Acknowledgments I’d like to firstly express my great appreciation to my advisor, Dr. Jiyoung Lee. She provides continuous guidance from the beginning of my admission no matter in my academic or personal life. Without her trusts and supports, I cannot finish my research and thesis. I appreciate all the help from my lab member Seungjun Lee, who answered my questions patiently, taught me when I made mistakes and comforted me when I was upset. It is great to work with him during two years. I also feel grateful to all my lab members and my committee members. They always provide essential help without hesitates and make me feel at home. Thanks for all the professors and friends. Your supports are the best gift. Last but not least, I appreciate all the supports from my family. They are my motivation to move forward, as well as my solid backup. Thank you for letting me choose my own life and supporting me all the time. v Vita September 2008 to June 2011 ........................Shanxi Experimental Secondary School August 2011 to July 2015 ..............................B.S. Biological Science, Nanjing University August 2015 to present .................................M.S. Department of Food Science and Technology, The Ohio State University Publications Lee J, Lee S, Jiang X. Cyanobacterial Toxins in Freshwater and Food: Important Sources of Exposure to Humans. Annual Review of Food Science and Technology. 2017 Apr;8(1). Fields of Study Major Field: Food Science and Technology vi Table of Contents Abstract ............................................................................................................................... ii Acknowledgments............................................................................................................... v Vita ..................................................................................................................................... vi Publications ........................................................................................................................ vi Fields of Study ................................................................................................................... vi Table of Contents .............................................................................................................. vii List of Tables ..................................................................................................................... xi List of Figures .................................................................................................................. xiii Chapter 1: literature review ................................................................................................ 1 1. Cyanotoxins: a growing concern for food safety and public health ............................ 1 1.1 Toxin-producing cyanobacteria: Harmful algal blooms ........................................ 1 1.1.1 Introduction of cyanobacteria ............................................................................. 1 1.1.2 Concerns about cyanobacterial blooms .............................................................. 2 1.1.3 Factors affecting bloom formation ..................................................................... 4 1.1.4 Monitoring of blooms ......................................................................................... 6 vii 1.2 Microcystins: the most widespread cyanotoxin ..................................................... 7 1.2.1 Introduction to microcystins ............................................................................... 7 1.2.2 Health impacts and guidelines of microcystins .................................................. 8 1.2.3 Exposure routes of microcystins for humans ................................................... 13 1.2.4 Effects of MCs on agriculture .......................................................................... 19 1.2.5 Quantification of microcystins ......................................................................... 21 1.2.6 Decontamination of MCs.................................................................................