Rapid Detection of Toxin-Producing Cyanobacteria

UNLV Theses, Dissertations, Professional Papers, and Capstones

5-1-2020

Rapid Detection of Toxin-Producing Cyanobacteria Rapid Detection of Toxin-Producing Cyanobacteria

Timothy Alba

Follow this and additional works at: https://digitalscholarship.unlv.edu/thesesdissertations

Part of the Microbiology Commons

Repository Citation Repository Citation Alba, Timothy, "Rapid Detection of Toxin-Producing Cyanobacteria" (2020). UNLV Theses, Dissertations, Professional Papers, and Capstones. 3857. http://dx.doi.org/10.34917/19412006

This Dissertation is protected by copyright and/or related rights. It has been brought to you by Digital Scholarship@UNLV with permission from the rights-holder(s). You are free to use this Dissertation in any way that is permitted by the copyright and related rights legislation that applies to your use. For other uses you need to obtain permission from the rights-holder(s) directly, unless additional rights are indicated by a Creative Commons license in the record and/or on the work itself. This Dissertation has been accepted for inclusion in UNLV Theses, Dissertations, Professional Papers, and Capstones by an authorized administrator of Digital Scholarship@UNLV. For more information, please contact [email protected].

http://library.unlv.edu/

http://library.unlv.edu/

https://digitalscholarship.unlv.edu/thesesdissertations

https://digitalscholarship.unlv.edu/thesesdissertations?utm_source=digitalscholarship.unlv.edu%2Fthesesdissertations%2F3857&utm_medium=PDF&utm_campaign=PDFCoverPages

http://network.bepress.com/hgg/discipline/48?utm_source=digitalscholarship.unlv.edu%2Fthesesdissertations%2F3857&utm_medium=PDF&utm_campaign=PDFCoverPages

http://dx.doi.org/10.34917/19412006

mailto:[email protected]

RAPID DETECTION OF TOXIN-PRODUCING CYANOBACTERIA

By

Timothy W. Alba

Bachelor of Science – Civil Engineering

University of Washington 2003

Master of Science – Civil Engineering University of Washington

2010

A dissertation submitted in partial fulfillment

of the requirements for the

Doctor of Philosophy – Biological Sciences

School of Life Sciences

College of Sciences The Graduate College

University of Nevada, Las Vegas May 2020

ii

Dissertation Approval

The Graduate College The University of Nevada, Las Vegas

May 1, 2020

This dissertation prepared by

Timothy W. Alba

entitled

Rapid Detection of Toxin-Producing Cyanobacteria

is approved in partial fulfillment of the requirements for the degree of

Doctor of Philosophy – Biological Sciences School of Life Sciences

Eduardo Robleto, Ph.D. Kathryn Hausbeck Korgan, Ph.D. Examination Committee Chair Graduate College Dean Mark Buttner, Ph.D. Examination Committee Member Kurt Regner, Ph.D. Examination Committee Member Christy Strong, Ph.D. Examination Committee Member Patricia Cruz, Ph.D. Graduate College Faculty Representative

iii

Abstract

Lake Mead provides drinking water to millions of people in Nevada, California, and Arizona. In

2015, the Southern Nevada Water Authority detected the cyanobacteria-produced toxin

microcystin in the lake for the very first time. This toxin is lethal in large doses, and in small

doses it causes a myriad of serious health effects. Detecting microcystin directly is a time-

consuming and expensive process that requires liquid chromatography tandem mass

spectrometry or immunological analyses, which require a full day or more to process samples. In

order to provide water managers with the methodology for a toxin monitoring plan, this work

developed a rapid and inexpensive protocol using real-time quantitative polymerase chain

reaction for the detection of microcystin-production genes that takes about 75 minutes to perform

by one person. Currently, water managers rely on microscopic identification for rapid detection

of microcystin-producing cyanobacteria, so this novel protocol is significant as an early indicator

for the presence of this toxin. The simulated algal blooms demonstrated that microcystin

production by cyanobacteria is nutrient-dependent, providing insight into additional water

quality monitoring tools. In addition, this work examined β-N-methylamino-L-alanine (BMAA),

a cyanobacteria-produced toxin associated with neurodegenerative disease. Although no BMAA

was detected in Lake Mead, simulated algal blooms suggest that the potential for BMAA

production in the lake is dependent on nutrient availability. The results of this study can be used

to detect toxin-producing cyanobacteria as part of a routine water quality monitoring program.

iv

Acknowledgements

Thank you to my committee for providing guidance and support throughout the duration of my project: Dr. Mark Buttner, Dr.Patricia Cruz, Dr. Kurt Regner, Dr. Eduardo Robleto, and Dr. Christy Strong.

I appreciate the efforts Dr. Penny Amy and Dr. Dennis Bazylinski made to support my progress as part of an earlier committee.

My very first class at UNLV was with Dr. Martin Schiller, and from that first lecture I was so inspired by his passion for biology, and that inspiration stayed with me throughout my time here on campus.

Dr. Helen Wing is always a pleasure to see on campus, she truly cares about students and it shows. It has been wonderful to be a grad student under her supervision as the graduate coordinator.

Dr. Tanviben Patel was so kind to voluntarily train me on any equipment or assay in the Emerging Diseases Laboratory or Pollen Laboratory at UNLV.

Keala Kiko at the College of Sciences has helped me immeasurably throughout my time at UNLV, and it is a real blessing to have him on campus.

I only had the chance to work directly with Nicole Espinoza for a couple semesters, but what a joy that was. Nicole is definitely one of my favorite people to just stop by her office and chat.

Dr. Javier Rodriguez helped me in some of my most difficult times, and his enthusiasm for science and dedication to students at UNLV are truly appreciated.

Casey Hall at the Genomics Center is incredible, I valued any chance I had to go there and train on a piece of equipment with her or talk through the details of a particular protocol.

Jamie Gilmore, Chantelle Martinez, and Theresa Trice at the Nevada State Public Health Laboratory were ready to help any chance I needed them, thank you.

Liz Detrick and Kristina Mihajlovski at the Emerging Diseases Laboratory were always so eager to help me with my project in any way they could, I greatly appreciate it.

Eric Wert, Brett Vanderford, and Katherine Greenstein at the Southern Nevada Water Authority helped me far beyond what I ever expected since they really believed in my project, and I cannot say thank you enough time to express my gratitude for their support.

Brittney Stipanov and Janie Holaday at SNWA were always in close contact to arrange for sample pickup, thanks for all those early mornings and chain of custody help.

Thanks to Dr. Jeremy Dodsworth and Dr. Senthil Murugapiran for your mentorship.

Special thanks to the 500+ students I had the privilege of teaching in BIOL 189, 251, and 351 labs. Sharing my love of microbiology with all of you was the highlight of each week for me.

v

Dedication

UNLV is an incredible school and I’ve been truly blessed to study here. I’ve been a Rebel about

as long as I can remember. I watched UNLV lose to Duke in the Final Four on TV at my

grandparent’s house in 1991 to miss out on the chance to win back-to-back NCAA basketball

national championships. That loss stung, even as a little kid. I’d be willing to bet I’m the only

current student that can say they watched that game. I’ve always seen UNLV as a place where

anything is possible, where you can make your dreams come true if you chase them with the

same tenacity as a Runnin’ Rebel.

I faced some challenges at UNLV that nearly made it impossible to achieve my dreams here.

Certainly every Ph.D. student faces tremendous obstacles, but I never expected to take 8.5 years

to graduate when I started in the Ph.D. program at UNLV. Despite my struggles, I know I’ll look

back on my studies at UNLV as some of the very best experiences of my life.

For my dedication I would like to highlight two professors at UNLV that are super heroes to me:

Dr. Mark Buttner and Dr. Eduardo Robleto

Without them, I would be long gone from UNLV, having transferred to a different school to

chase my dreams elsewhere. But that’s not what I wanted. I’m a Rebel. I fight for what’s right. I

didn’t want to go a different school. Because of these two very special people, I’m graduating

from UNLV and I’ve been offered an incredible job. Mark and Eduardo value student success,

and I owe them everything for that. They didn’t have to give all the time and energy it took to

help me be successful, but they did it when others wouldn’t, and that’s what makes them Rebels.

Thank you!

vi

Table of Contents

Abstract ........................................................................................................................................ iii

Acknowledgements ...................................................................................................................... iv

Dedication ..................................................................................................................................... v

List of Tables .............................................................................................................................. vii

List of Figures ............................................................................................................................ xiii

Chapter 1. Introduction ................................................................................................................. 1

Chapter 2. Materials and Methods .............................................................................................. 18

Chapter 3. Data and Results ........................................................................................................ 51

Chapter 4. Discussion and Conclusions ...................................................................................... 88

Appendix I – Growth Curves ...................................................................................................... 97

Appendix II – Microcystin Concentrations ............................................................................... 103

Appendix III – BMAA Concentrations..................................................................................... 111

Appendix IV – QPCR Results .................................................................................................. 114

Appendix V – Growth Media ................................................................................................... 118

Appendix VI – Culture Images ................................................................................................. 127

References ................................................................................................................................. 137

Curriculum Vitae ...................................................................................................................... 150

vii

List of Tables

Table 1. BMAA in freshwater cyanobacteria. (Image source: Cox et al., 2005) ......................... 11

Table 2. Cultures obtained from culture collections and associated growth media type. ............. 22

Table 3. Media recipes used to culture R. raciborskii compared to the media recipe recommended by ANACC (MLA). .............................................................................................. 32

Table 4. Detection of diverse cyanobacteria using universal cyanobacterial primers and probes (CYANO). SE – S. elongatus; FA – F. ambigua; NS – N. spumigena; LA – L. aesuarii; AF – A. flos-aquae; OL – O. lutea; NC – N. commune; C. diff – C. difficile (Negative control); NTC – No Template Control. Each abbreviated species name is also followed by either the number 0, -1, or -2. This system indicates if the DNA extract used was undiluted (100), diluted 1/10 (10-1), or diluted 1/100 (10-2). Ct = cycle at which fluorescence (amplicon) is 1st detected. ....................... 57

Table 5. Detection of new cultures using universal cyanobacterial primers and probes. All DNA extracts were diluted 1/10, except the C. difficile DNA was diluted 1/1000. ANT – Anabaena toxic; APH – Aphanizomenon; CR – Cylindrospermopsis raciborskii; MAT – Microcystis aeruginosa toxic; S – Synechococcus elongatus new culture; TRI – Trichodesmium erythraeum........................................................................................................................................................ 59

Table 6. DNA extraction buffer concentration effects on Ct. Each Sample Name starting with 1-, 2-, or 3- represents three different DNA extraction protocols, and Cts resulting from universal bacterial primers and probes according to each dilution. The sample used for DNA extraction was an M. aeruginosa pure culture (n=1). .................................................................................... 61

Table 7. Comparison of lake water + S. elongatus, molecular-grade water + S. elongatus, and S. elongatus pure culture dilutions, n=1 (LS-1 = Lake water + S. elongatus 10-1 dilution, MS-1 = Molecular-grade water + S. elongatus 10-1 dilution, S-1 = S. elongatus pure culture 10-1 dilution, Cdiff -3 = C. difficile 10-3 dilution, NTC = No Template Control). ............................................. 62

Table 8. Cyanobacteria detected in Lake Mead water using universal cyanobacterial primers and probes, n=1 (L-1 = lake water DNA 10-1 dilution, Cdiff-3 = C. difficile 10-3 dilution, NTC = No Template Control, S-1 = S. elongatus 10-1 dilution). ................................................................... 64

viii

Table 9. R. raciborskii primers and probes specificity, n=1 (NTC = No Template Control, RR-1 = R. raciborskii 10-1 dilution, MA-1 = M. aeruginosa 10-1 dilution, Cdiff-2 = C. difficile 10-2 dilution). ........................................................................................................................................ 64

Table 10. mcyD primer/probe comparison of toxin-producing and non-toxin-producing M. aeruginosa, n=1 (MA-1 = M. aeruginosa 10-1 dilution, MAT-1 = M. aeruginosa toxin-producing 10-1 dilution, Cdiff-3 = C. difficile 10-3 dilution, NTC = No Template Control). ..... 65

Table 11. mcyE primer/probes comparison of toxin-producing and non-toxin-producing M. aeruginosa, n=1 (MA-1 = M. aeruginosa 10-1 dilution, MAT-1 = M. aeruginosa toxin-producing 10-1 dilution, Cdiff-3 = C. difficile 10-3 dilution, NTC = No Template Control). ..... 66

Table 12. Optical density measurements corresponding with cell counts. ................................... 66

Table 13. Estimated number of cells present in each cell count culture DNA extraction. ........... 67

Table 14. Estimated mcyE/µL in the final DNA extract. .............................................................. 68

Table 15. Estimated cells per QPCR reaction well for the cell count culture. ............................. 68

Table 16. Cts associated with cell count (CC) dilutions. .............................................................. 68

Table 17. Toxin concentrations measured from M. aeruginosa pure culture used to inoculate other cultures. ................................................................................................................................ 70

Table 18. Comparison of estimated and actual toxin concentrations when inoculating a new culture. .......................................................................................................................................... 71

Table 19. 30-day toxin production, 200 mL BG-11 + 5 mL M. aeruginosa pure culture. ........... 72

Table 20. 60-day toxin concentrations from a 100 mL BG-11, 100 mL lake water, 5 mL M. aeruginosa culture. ....................................................................................................................... 73

Table 21. Toxin production in 200 mL lake water + 40 mL BG-11 + 5 mL M. aeruginosa. ...... 74

ix

Table 22. Toxin production in 200 mL lake water + 40 mL BG-11 + 5 mL M. aeruginosa repeated experiment. ..................................................................................................................... 75

Table 23. Comparison of pure culture (200mL BG-11) and reduced media (40mL BG-11) Ct values at approximately 60 days, assuming one copy of mcyE per cell. ...................................... 76

Table 24. Detection of Anatoxin-a in 200 mL BG-11 + 200 mL autoclaved lake water + 5 mL M. aeruginosa inoculum. ................................................................................................................... 76

Table 25. Difference in Ct between standard DNA extraction (EXTR) and freeze-thaw DNA extraction (HS). MAT10X is a 10X concentrated M. aeruginosa positive control, NTC is a No Template Control, n=1. ................................................................................................................. 83

Table 26. Difference in Ct between standard DNA extraction (10XExtr) and freeze-thaw DNA extraction (10XHS), n=1. .............................................................................................................. 84

Table 27. Blinded PCR results using 10X concentrated cells, compared to a 10X positive control (MAT10X = M. aeruginosa toxin-producing culture 10X concentrated positive control, NTC = No Template Control). .................................................................................................................. 85

Table 28. PCR results from a surface cell accumulation (Sample) compared with a M. aeruginosa toxin-producing pure culture (Positive Control). ....................................................... 86

Table 29. 10X concentrated results showing detection of a culture containing M. aeruginosa (MC+), compared to a culture lacking M. aeruginosa.................................................................. 87

Table 30. M. aeruginosa pure culture. .......................................................................................... 97

Table 31. M. aeruginosa 2nd pure culture. ................................................................................... 97

Table 32. M. aeruginosa 3rd pure culture..................................................................................... 98

Table 33. M. aeruginosa 4th pure culture. .................................................................................... 98

Table 34. M. aeruginosa grown in lake water. ............................................................................. 98

x

Table 35. M. aeruginosa 2nd culture grown in lake water. .......................................................... 98

Table 36. M. aeruginosa grown in autoclaved lake water. ........................................................... 99

Table 37. M. aeruginosa 2nd culture grown in autoclaved lake water. ........................................ 99

Table 38. M. aeruginosa grown in reduced media concentration. .............................................. 100

Table 39. M. aeruginosa 2nd culture grown in reduced media concentration. .......................... 100

Table 40. Growth of organisms naturally present in lake water. ................................................ 100

Table 41. No growth observed in lake water without growth media. ......................................... 101

Table 42. Growth of organisms in lake water with non-autoclaved fertilizer. ........................... 101

Table 43. Growth of R. raciborskii in modified combo media. .................................................. 101

Table 44. Growth of R. raciborskii in lake water amended with modified combo media.......... 101

Table 45. Growth of R. raciborskii in lake water. ...................................................................... 102

Table 46. Growth of organisms naturally present in lake water with modified combo media. .. 102

Table 47. Growth of R. raciborskii in autoclaved lake water amended with modified combo media. .......................................................................................................................................... 102

Table 48. R. raciborskii 2nd pure culture. .................................................................................. 102

Table 49. Lake water toxins ........................................................................................................ 103

Table 50. Low media concentration toxins. ................................................................................ 103

Table 51. Toxin production in lake water amended with growth media. ................................... 104

xi

Table 52. Toxin production in autoclaved lake water and lake water. ....................................... 104

Table 53. Pure culture M. aeruginosa toxin production. ............................................................ 105

Table 54. Second set of toxin data from M. aeruginosa pure culture. ........................................ 105

Table 55. Third set of toxin data from M. aeruginosa pure culture. ........................................... 106

Table 56. No toxins detected in low media concentration. ......................................................... 106

Table 57. Second set of samples showing no toxin detection in low media concentration. ....... 107

Table 58. Toxins produced by M. aeruginosa in autoclaved lake water amended with growth media. .......................................................................................................................................... 107

Table 59. High concentrations of toxins produced in M. aeruginosa pure culture. ................... 108

Table 60. Toxins produced by M. aeruginosa in lake water amended with growth media. ....... 108

Table 61. Toxins nearly undetectable in low media concentration. ........................................... 109

Table 62. No toxins detected in lake water, and nearly undetectable toxins in low media concentration. .............................................................................................................................. 109

Table 63. Toxins produced in lake water amended with growth media, and no toxins produced with very low media concentrations. .......................................................................................... 110

Table 64. No BMAA detected in R. raciborskii cultures in media or media + lake water. ........ 111

Table 65. No BMAA detected in 2nd set of R. raciborskii samples. ......................................... 111

Table 66. No BMAA detected in 3rd set of R. raciborskii samples. .......................................... 111

Table 67. BMAA detected in cyanobacterial cultures. ............................................................... 111

xii

Table 68. BMAA detected in 2nd set of cyanobacterial cultures. .............................................. 112

Table 69. No BMAA detected in tap water, bottled water, or lake water, but detected in seafood/cod (SF) and lake water with fertilizer added. .............................................................. 112

Table 70. Some BMAA detected in R. raciborskii cultures. ...................................................... 112

Table 71. BMAA detected in another R. raciborskii culture. ..................................................... 112

Table 72. No BMAA detected in a 2nd set of tap water, bottled water, lake water, and DI water samples. ....................................................................................................................................... 113

Table 73. BMAA detected in seafood samples. .......................................................................... 113

Table 74. BMAA detected in 2nd set of seafood samples. ......................................................... 113

Table 75. Near-limit of detection of mcyE genes. ...................................................................... 114

Table 76. Detection of mcyE genes from surface growth accumulation. ................................... 114

Table 77. Cell count standard curve, freeze/thaw method comparison, and blind experiment mcyE qPCR results. ..................................................................................................................... 115

Table 78. Freeze/thaw, blind experiment, and cell count dilution mcyE qPCR results. ............. 116

Table 79. Pure culture M. aeruginosa, reduced media concentration, and DNA extraction technique comparison mcyE gene qPCR. ................................................................................... 117

xiii

List of Figures

Figure 1. Microcystin general structure. (Image source: Mikalsen et al., 2003). ........................... 5

Figure 2. Chemical structure of BMAA. (Image source: Proctor et al., 2019). ............................. 9

Figure 3. BMAA incorporated into a protein chain. (Image source: Holtcamp, 2012). ................. 9

Figure 4. Visible wind-driven aggregation of biomass in the Las Vegas Boat Harbor Marina (Image source: Tietjen, 2015). ...................................................................................................... 14

Figure 5. Materials and Methods summary. ................................................................................. 19

Figure 6. Full-spectrum LED light mounted in the mirrored environmental chamber used in this study. ............................................................................................................................................. 24

Figure 7. 1000x photomicrograph of Microcystis aeruginosa LB 2388. ..................................... 25

Figure 8. 1000x magnification photomicrograph of Oscillatoria lutea var. contorta UTEX LB 390................................................................................................................................................. 25

Figure 9. A single filament of Nodularia spumigena B 2091 at 1000x magnification. ............... 26

Figure 10. 1000x magnification photomicrograph of Lyngbya aestuarii UTEX LB 2515. The arrow points to an individual cell within a sheath of multiple cells. ............................................ 27

Figure 11. 400x magnification photomicrograph of Nostoc commune B 1621. ........................... 28

Figure 12. Synechococcus elongatus 2973 cells at 1000x magnification in pure culture. ............ 29

Figure 13. Example standard curve used to determine BMAA concentrations of environmental samples from absorbance data (Source: Southern Nevada Water Authority, unpublished). ........ 44

Figure 14. Buoyant M. aeruginosa (Kützing) cells accumulated at the water surface. ................ 46

xiv

Figure 15. MasterMix preparation in a clean work station. .......................................................... 48

Figure 16. The 96-well plate prepped for loading into the 7900HT. ............................................ 49

Figure 17. 30-day growth curves associated with standard pure culture replicates. Cultures 1 through 4 are all 200 mL BG-11 + 5 mL Microcystis pure cultures............................................. 52

Figure 18. 30-day growth curves associated with standard pure culture replicates. Cultures 1 through 4 are all 200 mL BG-11 + 200 mL autoclaved lake water + 5 mL Microcystis pure cultures. ......................................................................................................................................... 53

Figure 19. Cultures mimicking a CyanoHAB in Lake Mead. ...................................................... 54

Figure 20. Culture with media reduced to 40 mL BG-11. ............................................................ 55

Figure 21. Lake Water + BG-11 media growth curve. ................................................................. 56

Figure 22. Estimated 30-day growth curve relating cell count and OD600. .................................. 67

Figure 23. A standard curve relating # cells in a QPCR reaction well and resulting Ct............... 69

Figure 24. Summary of a comprehensive plan for routine monitoring, sampling of visible surface accumulated growth, and lab monitoring...................................................................................... 95

Figure 25. Standard 200 mL BG-11 + 5 mL M. aeruginosa Kützing pure culture. ................... 127

Figure 26. Typical 200 mL BG-11 + 200 mL autoclaved lake water + 5 mL M. aeruginosa culture. ........................................................................................................................................ 127

Figure 27. 100 mL Lake Water + 100 mL BG-11 + 5 mL M. aeruginosa. ................................ 128

Figure 28. 200 mL Lake Water + 40 mL BG-11 + 5 mL M. aeruginosa. .................................. 128

Figure 29. 100 mL Lake Water + 40 mL BG-11 + 5 mL M. aeruginosa culture results in growth that is not buoyant and settles to the bottom of the flask. The green color at the surface of the water is an optical illusion, reflected from the growth below. .................................................... 129

xv

Figure 30. 50 mL lake water + 50 mL BG-11 media, no Microcystis added. ............................ 129

Figure 31. 400x photo of cells naturally present in Lake Mead water and cultivated using BG-11 media. .......................................................................................................................................... 130

Figure 32. Cells grown from organisms naturally present in Lake Mead water are not buoyant...................................................................................................................................................... 130

Figure 33. No growth in autoclaved lake water. ......................................................................... 131

Figure 34. No growth in autoclaved lake water amended with BG-11. ..................................... 131

Figure 35. No growth in lake water with no media added. ......................................................... 132

Figure 36. M. aeruginosa grows in autoclaved lake water. ........................................................ 132

Figure 37. M. aeruginosa grows at very low media concentration (1 mL BG-11). ................... 133

Figure 38. Fertilizer used to mimic a source of stormwater pollution in Lake Mead................. 133

Figure 39. Nitrogen and phosphorus composition of the fertilizer used in this study. ............... 134

Figure 40. 1 g of non-autoclaved fertilizer promoted growth in Lake Mead water. ................... 134

Figure 41. The fertilizer in the left flask was not autoclaved prior to adding it to Lake Mead water. The fertilizer in the right flask was autoclaved, but it solidified, and digging it out damaged the fertilizer pellets. The fertilizer in the center flask was autoclaved, and did not produce growth. .......................................................................................................................... 135

Figure 42. R. raciborskii pure cultures turn orange over time. ................................................... 135

Figure 43. R. raciborskii culture is green before turning orange over time................................ 136

Figure 44. Color difference between lake water cultured in Combo and BG-11 media. ........... 136

1

Chapter 1. Introduction 1.1. Cyanobacteria

Cyanobacteria are ancient, with fossil records indicating that ancestral cells existed on Earth

around 3.5 billion years ago, making them among the oldest and most important organisms due to

their role as primary producers (Schopf, 1993; Olson, 2006; Tomitani et al., 2006; Whitton, 2012).

Cyanobacteria have evolved to thrive in every ecological niche on Earth, to include terrestrial,

aquatic, and subsurface habitats (Paerl and Huisman, 2009; Rastogi et al., 2014). Cyanobacteria

thrive in both freshwater lakes and the hyperoligotrophic open ocean and can be found living

psychrotrophic lifestyles near 0°C, in hot springs at 73°C, and everywhere in between (Whitton

and Potts, 2012). Cyanobacteria also have amazing morphological diversity, ranging from

unicellular lifestyles to multi-cellular filaments and trichomes (Uyeda et al., 2016). Although more

than 2600 species of cyanobacteria are known, it is thought that there are large numbers of

unknown species as well (Nabout et al., 2013).

Excessive growth of cyanobacteria can result in a visible accumulation of cells, commonly

referred to as a harmful algal bloom (HAB) or more specifically, a CyanoHAB (Huisman et al.,

2005). Hypereutrophication, or an over-abundance of nutrients such as nitrogen and phosphorus,

contributes to the density and frequency of algal blooms (Dodds et al., 2009). Also, climate

change and the associated increases in temperatures and droughts affect the occurrence and

intensity of algal blooms (Paerl and Huisman, 2009; Balbus et al., 2013). All these antecedents

have led to an increased concern about the global environmental impacts of CyanoHABs and

better monitoring for their occurrence (Davis et al., 2009; Zhang et al., 2012; Scholz et al., 2017;

Griffith and Gobler, 2019). Current monitoring strategies employ microscopic cell counts and

satellite imagery, with both approaches having significant limitations (Recknagel et al., 1997,

2

Downing et al., 2001; Allen et al., 2008). Therefore, there is a need for standardized CyanoHAB

monitoring strategies that go beyond these current approaches.

Cyanobacteria produce secondary metabolites that can benefit energy, pharmaceutical, and

agricultural industries (Radau et al., 2003; Ersmark et al., 2008; Vijayakumar and Menakha, 2015).

Although biofuel production has recently relied more heavily on eukaryotic algae (Ajjawi et al.,

2017), cyanobacteria have shown tremendous potential for the development of drugs to treat high

blood pressure, myocardial infarction, stroke, cancers, and viral infections (Radau et al., 2003;

Borgono and Diamandis, 2004; Ersmark et al., 2008). However, some cyanobacterial byproducts

are toxic to humans and aquatic life (Falconer, 1998; Chorus and Bartram, 1999; Kuiper-Goodman

et al., 1999; Carmichael et al., 2001; Stewart et al., 2008; Zegura et al., 2011; Mowe et al., 2015;

Drobac et al., 2016). These are commonly referred to as cyanotoxins.

1.2. Cyanotoxins

The factors that contribute to the production of cyanotoxins are not well understood (Heisler et

al., 2008; Pick 2016). Routes of exposure to cyanotoxins include entering through the skin while

swimming, through inhalation when aerosolized, or consumption in drinking water and seafood.

There are currently no national standards or regulations for measuring cyanotoxin concentrations

in drinking water or food. It is well established in the literature that cyanotoxins are neurotoxic,

hepatotoxic, and tumor promoting in humans, but the concentrations and distribution of these

toxins in freshwater and marine habitats that lead to toxicity are not well defined. For example,

limitations of sampling frequency have been problematic in previous studies (Briand et al. 2009;

Guedes et al., 2014). Sampling frequency limitations are specifically due to the time and cost

associated with detecting cyanotoxin molecules directly.

3

To compound the sampling problem, cyanobacteria produce hundreds of different potentially

toxic or bioactive peptides (TBPs), and it is unknown if most of these microviridins, microginins,

cyclamides, cyanopeptolins, cryptophycins, aeruginosins, and anabaenopeptins are toxic (Welker

and von Dohren, 2006; Sivonen et al., 2010; Chlipala et al., 2011). Also, novel peptides

produced by this phylum and their toxicity remain uncharacterized (Welker et al., 2004).

Cyanotoxins are not currently measured and regulated because it is not cost effective to directly

measure cyanotoxin concentrations using enzyme-linked immunosorbent assay (ELISA) or liquid

chromatography tandem mass spectrometry (LC-MS/MS) on a routine basis at water quality

laboratories around the world. In addition, next-generation sequencing approaches to evaluate

environmental samples are still too complex and time-consuming to be performed rapidly in

municipal water quality laboratories. Despite the fact that there are hundreds of different TBPs

produced by cyanobacteria, there are only a few well-studied types of cyanotoxins. These are:

Microcystin (Carmichael et al., 1975), Cylindrospermopsin (Devlin et al., 1977), Anatoxin

(Carmichael and Gorham, 1978), and Saxitoxin (Jackim and Gentile, 1968).

In 2015, microcystin-producing algal blooms occurred in Lake Mead. This dissertation focuses on

cyanobacteria that produce microcystins, and on the lesser-studied toxin β-N-methylamino-L-

alanine (BMAA). Although microcystin receives the majority of the focus of this study due to

having been detected in Lake Mead previously, an objective of this study with regards to BMAA

was to determine if it is present in Lake Mead and in Las Vegas drinking water.

1.2.1. Microcystin

The most commonly studied cyanotoxins are microcystins (World Health Organization, 2003).

Microcystins are mostly intracellular, with < 30% released extracellularly (Graham et al., 2010).

4

Environmental factors including trace elements and light availability contribute to microcystin

production (Kaebernick et al., 2000; Tonk et al., 2005; Sevilla et al., 2008).

Microcystins are cyclic heptapeptides and have the following general structure:

D-Ala1 -X2 -D-MeAsp3 - Z4 -Adda-Arg5 -D-Glu6 -Mdha7

- Amino acid positions at X2 are variable

- Amino acid positions at Z4 are either glutamic acid or glutamine

- D-MeAsp = D-erythro-β-methylaspartic acid

- Adda = (2 S, 3 S, 8 S, 9 S)- 3-amino-9-methoxy-2–6–8-trimethyl 10-phenyldeca-4,6-

dienoic acid

- Mdha = N-methyldehydroalanine

The most studied microcystin is X2 = Leucine (L) and Z4 = Arginine (R), or commonly referred

to as MC-LR. Mcy genes control the non-ribosomal synthesis of microcystins through the

products of ten open reading frames that encode polyketide synthases and peptide synthases

(Dittmann et al., 1997; Kaebernick et al., 2002). Figure 1 illustrates how McyA through McyJ

synthesize microcystin.

5

Figure 1. Microcystin general structure. (Image source: Mikalsen et al., 2003).

A full list of microcystin variants includes more than 240, with additional variants still being

found (Spoof and Catherine, 2017). Due to the number of variants in the structures, the toxicity

can vary with several different toxicokinetic profiles (Bouaïcha et al., 2019). The focus on MC-

LR has led to the incorrect assumption that the other variants are of no health concern (Testai et

al., 2016). The most studied variants among the known 240+ are MC-LR, MC-RR, and MC-YR.

Some of these variants have been shown to have an array of different health impacts on humans.

Microcystins have been shown to impact the liver and other tissues through inhibition of

serine/threonine protein phosphatases 1 and 2a (MacKintosh et al., 1990; Fischer et al., 2005;

Feurstein et al., 2009). Microcystins induce cell lysis in liver cells at a dose above 40 µg/kg

6

(Fawell et al., 1999). It is speculated that microcystins can cross the blood-brain barrier and

inflict neurotoxicity (Fischer et al., 2005; Feurstein et al., 2009; Li et al., 2014). Male

reproductive organs have been impacted by microcystins in rodent studies (Chen et al.,. 2011).

Microcystins are associated with carcinogenesis and disrupt cell signaling and cell growth

(Nishiwaki-Matsushima et al., 1992; Falconer and Humpage, 1996; Humpage et al., 2000;

Gehringer, 2004; Agudo et al., 2010). Chronic effects of microcystin exposure may contribute to

colorectal and liver cancer (Fleming et al., 2002; Lun et al., 2002; Zhou et al., 2002; Li et al.,

2011). From liver damage to cancer, it is clear why finding microcystin in Lake Mead, the

drinking water source for millions of people, represents a major risk to public health. Despite

these concerns, it is important to note that no federal guidelines exist for cyanotoxins within the

Safe Drinking Water Act (SDWA) or the Clean Water Act (CWA) (U.S., 1996). However,

Microcystin-LR is listed on the EPA’s Candidate Contaminant List (CCL) (EPA, 2016).

Microcystins are known to be produced by some species within the following genera:

Microcystis, Nodularia, Anabaena, Oscillatoria, Nostoc, Fischerella, Planktothrix, and

Gloeotricia. The species that was associated with microcystin production in Lake Mead in 2015

was Microcystis aeruginosa Kützing; therefore, Microcystis was the focus of this study.

Microcystis presence, surface water temperature, nutrient concentrations, lake mixing and wind

speed are all parameters of concern in microcystin production (Jacoby et al., 2000; Kotak et al.,

2000; Francy et al., 2016; Harris and Graham, 2017). Microcystin concentrations exceeded 1

µg/L in Toledo, Ohio in August 2014, and residents were directed to refrain from the use of tap

water (Wynne and Stumpf, 2015), which is a massive burden on the public and a serious

directive. To compound the problem for water managers, microcystin can remain in water for

weeks and long after a CyanoHAB. Microcystin, unlike other toxins, is highly resistant to

7

degradation (Zastepa et al., 2014), and is still active even after boiling treatment (Zamyadi et al.,

2012). This observation explains why Toledo, Ohio residents were directed to refrain from using

the water at all. Microcystin-LR recommendations are in the 0.3 µg/L (children younger than

pre-school age) to 1 µg/L (older children and adults) range for drinking water, and for

consumption in food, the adult tolerable daily intake (TDI) is 0.04 µg/kg body weight/day

(WHO, 2003, Chorus and Bartram, 1999). Exposure to the toxin during recreational activities

should be avoided when MC-LR concentrations are greater than 4 µg/L. No guidelines have been

set for other microcystin variants, which underlines the need for better understanding the health

impacts of 240+ known variants of this toxin, and only one is mentioned by the WHO and the

EPA. Microcystins are not just a concern in bodies of freshwater; CyanoHABs are a major

problem in the Baltic Sea, Gulf of Finland, and Gulf of Riga (Mazur and Plinski, 2003; Purvina

et al., 2008; Fewer et al., 2009; Semenova et al., 2017). Both Microcystis aeruginosa and

Planktothrix agardhii have been identified as potential microcystin producers in the brackish

waters of the Gulf of Finland (Chernova et al., 2018).

Some studies have directly measured the concentrations of other microcystin variants. In the

Koka Reservoir in Ethiopia, MC-LR was 815 µg/L, MC-YR was 466 µg/L, and MC-RR was 265

µg/L, which corresponded with extracellular concentrations of 20 µg/L MC-LR, 6.13 µg/L MC-

YR, and 1.27 µg/L MC-RR, respectively, indicating that the vast majority of microcystins are

maintained intracellularly (Tilahun et al., 2019). Water treatment methods require that the cells

do not get disturbed in a way that can cause them to release the intracellular toxins (Hawkins et

al., 1985; de Figueiredo et al., 2004). This is because cyanotoxins are much more difficult to

remove from drinking water by conventional water treatment processes once they have been

released from Microcystis cells (de Figueiredo et al., 2004).

8

Nitrogen and phosphorus nutritional levels in the cell have very significant roles in microcystin

production (Orihel et al., 2012; Scott et al., 2013; Jacoby et al., 2015). More specifically, nitrate

has been suggested to be a key determining factor because Microcystis species do not fix

atmospheric nitrogen (Tilahun et al., 2019). This focus on the role of nitrogen is a departure from

common assumptions about cyanobacteria due to the prevalence of nitrogen fixation within the

phylum. Although difficult to prove, it has been suggested that Microcystis is successful in

outcompeting other cyanobacteria in low phosphorus, high nitrogen conditions (Paerl and Otten,

2013). In addition, higher temperatures have been linked with increased toxicity in Microcystis

(Davis et al., 2009). The specific causes of high microcystin production are unknown (Scholz et

al., 2017; Harris and Graham, 2017).

1.2.2. β-N-Methylamino-L-Alanine (BMAA)

It is becoming increasingly evident that microbial metabolites have a role in neurodegenerative

diseases, particularly dementia disorders such as Alzheimer’s Disease (AD) (Murch et al., 2004;

Pablo et al., 2009; Holmes and Cotterell, 2009; Scott and Downing, 2018), Amylotrophic Lateral

Sclerosis (ALS) (Pablo et al., 2009; Scott and Downing, 2018), and Amylotrophic Lateral

Sclerosis/Parkinsonism Dementia Complex (ALS/PDC) (Zimmerman, 1945; Arnold et al., 1953;

Kurland and Bulder, 1954; Duncan et al., 1990; Cox and Sacks, 2002; Murch et al., 2004), or

perhaps diseases that closely mimic AD, ALS, and Parkinson’s.

β-N-methylamino-L-alanine (BMAA) is a non-canonical polar amino acid with a reactive

nitrogen group. The structure is shown in Figure 2.

9

Figure 2. Chemical structure of BMAA. (Image source: Proctor et al., 2019).

A 2005 article in the Proceedings of the National Academy of Sciences indicated that among 30

different cyanobacterial species tested in the laboratory, 95% produce BMAA (Cox et al., 2005).

BMAA production has been suggested to be induced by nitrogen deprivation, which may explain

why it is a common metabolite in all cyanobacteria (Downing et al., 2011; Scott et al., 2014).

Researchers speculate that BMAA binds to serine transfer RNA and is incorporated into proteins,

causing protein misfolding, as shown in Figure 3 (Rodgers and Dunlop, 2011).

Figure 3. BMAA incorporated into a protein chain. (Image source: Holtcamp, 2012).

The first hints of implications of BMAA for global health came after World War II, when

surveys were made of previously occupied regions, including Guam. A survey identified a

10

serious epidemic of ALS/PDC among the Chamorro Indians that remains controversial to this

day (Zimmerman, 1945). Chamorro Indians have lived in Guam for hundreds of years, with a

population as high as 100,000 people around the 16th or 17th centuries having been reduced to

around 10,000 during a 1901 census. Although ALS generally affects about 1 or 2 people per

100,000, the rate among the Chamorro people was about 100 times higher than rates found

elsewhere in the world (Kurland and Mulder, 1954). This sparked considerable attention from

physicians working in Guam, and that attention was quickly placed on the unique Chamorro diet.

The primary dietary source of carbohydrates for Chamorro people were cycad seeds, produced

by the indigenous Cycas micronesica. Not only did the Chamorro people use the seeds to

produce flour, they also consumed the meat of pigs and flying foxes that consume cycad seeds.

Researchers discovered that symbiotic cyanobacteria reside in specialized coralloid roots of the

cycad, and that these cyanobacteria produce the neurotoxic non-protein amino acid BMAA that

is deposited in cycad seeds and biomagnifies in animals that forage for cycad seeds. The

Chamorro diet was adjusted based on these warnings, and the rates of ALS have since returned

to normal levels.

This problem is not isolated to a small population or a single incident that happened decades ago.

Finland, a highly developed nation, has the highest dementia mortality rate in the world;

correspondingly, the average diet in this country includes over 70 pounds of fish per year, with

fish being candidates for BMAA bioaccumulation (Eiser, 2017). Other locations in which

BMAA is suspected to be in seafood are Florida (Brand, 2009; Mondo et al., 2012), Scandinavia

(Jonasson et al., 2010; Jiang et al., 2014; Salomonsson et al., 2015), and the Chesapeake River

(Brand et al., 2010). One study concludes that the sale and consumption of fish that have not

been tested for BMAA must be reconsidered (Scott et al., 2018). BMAA and other neurotoxins

11

consumed in food and drinking water might have a major impact on global incidence of

neurodegenerative disease, which has serious consequences for human health worldwide.

Current environmental regulations do not protect the public from this toxin. As an example, there

are only three cyanotoxins on the EPA Contaminant Candidate List: Microcystin-LR,

Cylindrospermopsin, and Anatoxin-a. The absence of other toxins, including BMAA, is a major

concern for public health.

Although the main objective of the BMAA portion of this study was to determine if this toxin is

present in Lake Mead and Las Vegas tap water, Table 1 demonstrates a secondary objective for

BMAA, which was to determine if a freshwater BMAA-producing culture can be grown in the

laboratory to better understand rates of BMAA production.

Table 1. BMAA in freshwater cyanobacteria. (Image source: Cox et al., 2005)

The cyanobacterial species Cylindrospermopsis raciborskii CR3 is shown to produce 6492 µg/g

of free BMAA in freshwater, far surpassing all other freshwater origins. As a secondary

objective, this study will attempt to replicate this level of BMAA production. As a third

12

objective, this study aimed to determine if BMAA was present in seafood purchased from a local

supermarket.

It is important to note that BMAA is a small molecule that is difficult to detect, which makes

detection more complex than detection of molecules with larger molecular weight. Standard

methods include liquid chromatography tandem mass spectrometry (LC-MS/MS), but even these

standard protocols may be ineffective because BMAA binds to protein and surfaces, and is

removed during sample preparation, which results in inaccurate estimation of the concentration

of BMAA in a sample. BMAA was measured using ELISA in this study in order to avoid the

problems associated with BMAA binding to surfaces in the LC-MS/MS protocol.

1.3. Lake Mead

In terms of storage volume, Lake Mead is the largest freshwater reservoir in the U.S. (Holdren

and Turner, 2010). More than 30 million people in Nevada, California, and Arizona consume

drinking water from Lake Mead, and more than 8 million people visit the lake recreationally

each year. The lake receives inflow from the Colorado River, with some marginal inputs from

the Virgin and Muddy Rivers, as well as the Las Vegas Wash. The Colorado River passes

through Hoover Dam as outflow. The Colorado River generally lacks pollution upstream of Lake

Mead, and therefore, the vast majority of pollutants present in Lake Mead originate from the Las

Vegas Wash, which is 100% composed of wastewater treatment plant effluent and untreated

stormwater runoff from Las Vegas (Snyder et al., 1999; LaBounty and Burns, 2005). The

physicochemical characteristics of Lake Mead, namely pH, temperature, and dissolved oxygen

are well described (LaBounty and Horn, 1997; LaBounty and Burns, 2005; Holdren and Turner,

2010). Lake Mead is described generally as a warm monomictic lake, with occasional variations

on thermal stratification and mixing. This generally means that during cold months the water

13

temperature is roughly the same throughout the entire water column (11-12°C), with no surface

freezing, which allows for one period of mixing all winter. Aside from this one mixing period,

the lake then undergoes thermal stratification throughout the rest of the year, where the surface

water becomes warm and there is no mixing with the colder, deeper water. As noted earlier,

thermal stratification throughout most of the year, and a very short mixing period can occur.

Lake Mead has experienced an extended period of drought since the year 2000, resulting in

decreased water surface elevations and increased water temperatures. Droughts, increasing water

temperatures, higher nutrient concentrations from stormwater inputs, and thermal stratification

during seasons that typically experience mixing will result in more harmful algal blooms

(Mosley, 2005; Paerl and Huisman, 2008). High phosphorus levels prior to 2002 have been

associated with a long history of non-toxic algal blooms at Lake Mead (LaBounty and Burns,

2005). Chlorophyll a has been used as a water quality characteristic in the past to determine toxic

levels of cyanotoxins, but this method has come into question because Lake Mead has abundant

non-nuisance, non-toxic bloom-forming cyanobacterial species (Rosen et al., 2012). Therefore,

chlorophyll a was not a parameter of interest in this study. Wastewater treatment processes were

improved to limit nutrient discharges following a 2001 algal bloom (LaBounty and Burns, 2007).

A notable change in Lake Mead was the discovery of microcystin and Microcystis aeruginosa

(Kützing) blooms, possibly caused by drought conditions, for the first time in the lake in 2015.

Interestingly, Microcystis was reported before in Lake Mead, but not microcystin (Rosen et al.,

2012). Low snowpack from a warm winter preceded the 2015 bloom, only exacerbating the

drought that had been lowering Lake Mead’s water surface elevation for the past 15 years. On

March 11, 2015, the City of Las Vegas and the Southern Nevada Water Authority were notified

14

of a visible wind-driven aggregation of cell growth by staff at the USGS and National Park

Service (Figure 4).

Figure 4. Visible wind-driven aggregation of biomass in the Las Vegas Boat Harbor Marina (Image source: Tietjen, 2015).

Based on microscopic identification of Microcystis cells, health advisories were issued for the

Lake Mead National Recreation Area. Samples taken from the surface aggregation of cells were

later examined using LC/MS-MS and the toxin microcystin was detected in the range of 84 to

470 µg/L (Tietjen, 2015). Lake Mead has not experienced a CyanoHAB during the duration of

this study, making it impossible to obtain samples for testing toxin-detection protocols.

Therefore, this study was centered on growing toxic CyanoHABs in the laboratory. Samples of

15

Lake Mead water were generously provided by the Southern Nevada Water Authority enabling

the cultivation of simulated Lake Mead CyanoHABs in the laboratory. In order to provide the

Southern Nevada Water Authority with the methodology for rapidly and inexpensively

monitoring Lake Mead with an early-warning system for toxin-producing cyanobacteria, the

following assay was used, as described in Section 1.4.

1.4. TaqMan QPCR Background

Serious questions remain in the understanding of CyanoHABs:

- Is there a threshold of concern when detecting cyanotoxins?

- What actions need to be taken when cyanotoxins are detected?

- Does the detection of a toxin mean that a water system is impaired?

Of all the questions that have been posed by water quality managers around the world regarding

cyanotoxins, this study focused on one particular issue:

- Can QPCR be used as an early warning system for cyanotoxins?

Quantitative polymerase chain reaction (QPCR) is used to count the number of target genes in a

sample. When oligonucleotide primers are added to a DNA template in a standard thermocycling

protocol, primers anneal to target sites, but an oligonucleotide fluorescently-labeled probe is also

present in the reaction when performing a TaqMan assay (Holland et al., 1991). The probe is

labeled with a reporter and a quencher. When the quencher is in close proximity to the reporter,

the reporter does not emit fluorescence. When DNA polymerase activity extends the forward

primer, the 5’ to 3’ exonuclease activity destroys the probe and releases the reporter. When the

quencher is no longer absorbing the emission from the reporter when the two are separated from

the probe, the reporter gives off detectable fluorescence. For each amplicon that is synthesized

16

throughout a standard 40-cycle TaqMan QPCR reaction, the accumulation of fluorescence is

detected by the QPCR platform of choice (e.g. 7900HT, QuantStudio, etc.). When the

fluorescence is detected above a “background” level, the cycle associated with this level of

detection is commonly referred to as the concentration threshold (Ct). TaqMan QPCR was used

to determine the Ct associated with amplifying microcystin-production genes and measured the

number of Microcystis aeruginosa cells that are present in that sample. Microcystin-production

genes have been used in PCR protocols as monitoring tools (Baker et al., 2001; Nonneman and

Zimba, 2002; Pan et al., 2002; Jungblut and Neilan, 2006). The mcyE protocol is useful in

detecting all microcystin producers, not just Microcystis, to include Anabaena and Planktothrix

species (Jungblut and Neilan, 2006). There was a correlation found between mcyE gene copy

number and microcystin concentration (Vaitomaa et al., 2003). There is a possible correlation

between cell density and microcystin synthesis (Wood et al., 2012). In contrast, some studies

suggest there is no link between mcy genes and microcystin concentrations (Guedes et al., 2014;

Beversdorf et al., 2015). Reports showed that detection of mcy genes may be inadequate as an

indicator of microcystin production (Lu et al., 2020). Clearly, the literature is controversial about

the uses of QPCR as an early warning system for cyanotoxins, which leads to the overall

objectives of this study.

1.5 Project Objectives

This project was anchored in the establishment of simulated toxic CyanoHABs in the laboratory

to supply samples for study, due to the current absence of CyanoHABs in Lake Mead. The key to

a simulated bloom was to grow toxic cyanobacteria in water from Lake Mead, so it was

necessary to work with the Southern Nevada Water Authority to obtain these lake water samples.

It was also necessary to obtain a light source, growth media, and toxin-producing cyanobacterial

17

cultures from existing culture collections. Once these simulated blooms were started in the

laboratory, this project focused on the following five objectives:

1. Establish a surrogate (simulation) system to grow M. aeruginosa in Lake Mead conditions.

2. Use QPCR to quantify the density of M. aeruginosa from the surrogate system.

3. Quantify toxin production from the surrogate system.

4. Study the production of other cyanotoxins.

5. Develop a protocol for water quality control/management.

18

Chapter 2. Materials and Methods

The methods used to achieve all five objectives included the following major tasks:

- Order cyanobacterial cultures and growth media from culture collections

- Grow cyanobacterial cultures in the laboratory using an LED light

- Obtain water from Lake Mead

- Grow surrogate Lake Mead algal blooms in the laboratory

- Sample simulated blooms for OD600 optical cell density

- Save OD600 samples for DNA extraction and toxin measurement

- Use a cell counter to determine how OD600 equates to cells per mL

- Use TaqMan primers and probes to target cyanobacterial genes of interest

- Extract DNA from samples

- Perform QPCR to determine Ct values, using known concentration cultures as standards

- Determine toxin concentrations using LC-MS/MS and ELISA

19

Figure 5. Materials and Methods summary.

2.1. Culture Conditions

2.1.1. Culture Equipment

An important factor in culturing cyanobacteria in the laboratory is the selection of a light source.

Cool white fluorescent lamps are the most common light source used in the literature, but LEDs

(light emitting diodes) are increasing in use in recent years, and provide a broader spectrum of

light than fluorescent bulbs. In this study, light was provided using a full-spectrum dimmable

150W cold red (620-630nm) to blue (460-465 nm) LED (type SMD2835) light array (EmaSun,

China), with dimensions of 30 cm x 30 cm x 1 cm. Twelve hour light/dark cycles were controlled

using a Purple Reign Dual Outlet grow light meter (Apollo Horticulture, USA). In order to mount

20

the grow light, a VWR Model #1575 benchtop incubator shaker with interior dimensions of

19”x19.25”x23.5” was used, which includes a shelf for mounting the LED light. The inside of the

incubator is lined on all sides with mirrors. A particular limitation of this incubator model is the

inability to cool the incubator to temperatures below ambient, as the range is ambient to 37°C. In

order to lower the temperature inside the incubator shaker, ice packs were placed inside the

incubator on a daily basis, dropping the temperature below ambient before eventually returning to

ambient temperature after the ice packs thawed. In some laboratories, cyanobacteria are grown

inside temperature-controlled environmental chambers with a constant temperature of 20°C, for

example. In this study, the temperature cycled from approximately 30°C to 20°C. This fluctuation

was determined to be acceptable due to this temperature range somewhat matching that of the

range of lake water temperatures in the summer bloom months averaging around 20°C in May and

moving up to a high of around 30°C in July/August. A growth temperature between 20°C and

30°C mimics the environmental conditions found in blooms and is not detrimental to the growth

of cyanobacteria in the laboratory. The dimmable light was operated at maximum intensity. Light

intensity was measured using an LT40 LED light meter (ExTech Instruments, Nashua, NH), and

temperature and humidity inside the incubator were recorded using a thermometer/humidity gauge

(AcuRite, Lake Geneva, WI). Cultures were grown in sterile Erlenmeyer flasks capped with

aluminum foil. Flasks containing growth media were inoculated using aseptic techniques with a

sterile single-use pipette inside of a Purifier Class II Biological Safety Cabinet with dual laminar

flow and HEPA filtration (LABCONCO, Kansas City, MO). Cultures were grown with continuous

agitation at 60 rpm in the benchtop incubator shaker.

21

2.1.2. Culture Methods

With regards to the task of growing simulated Lake Mead algae blooms in the laboratory, it is

important to note that there is a resource limitation due to the use of a single incubator with a single

LED light. Ideally, multiple environmental chambers would be available, where multiple LED

lights could be installed to grow a larger quantity of simulated blooms using several more cultures,

increasing the statistical significance of the results in this study. However, these resources were

not available in this project. Given those recognized constraints, culture conditions were

established according to the following concepts. First, the standard bottle-set for growing

Microcystis aeruginosa in the laboratory according to techniques established by the Southern

Nevada Water Authority in their studies is to inoculate 5 mL of an existing M. aeruginosa culture

into 200 mL of growth media using aseptic techniques to avoid bacterial or fungal contamination

of a pure culture. As a complementary experiment to this standard culture, 100 mL of growth

media mixed with 100 mL of Lake Mead water were inoculated with 5 mL of a Microcystis

aeruginosa pure culture, resulting in the same total volume (200 mL). Recognizing that there is

half of the media available in this culture, a further experiment was set up with 200 mL growth

media, 200 mL Lake Mead water, and 5 mL of a Microcystis aeruginosa pure culture. In order to

assess the impact of the microorganisms present in the Lake Mead water, another culture

containing 200 mL growth media, 200 mL autoclaved Lake Mead water, and 5 mL of a Microcystis

aeruginosa pure culture was established. In order to cultivate only the microorganisms present in

the lake water, a control culture was established with 100 mL Lake Mead water and 100 mL growth

media only. Additionally, a culture with reduced media concentration was made, with 200 mL

Lake Mead water, 40 mL growth media, and 5 mL of a Microcystis aeruginosa pure culture.

Additional controls including lake water only, autoclaved lake water only, and autoclaved lake

22

water plus growth media complemented the previously mentioned cultures. Similar approaches

were applied to cultures inoculated with Cylindrospermopsis raciborskii to assess BMAA

production, due to the concerns addressed in Section 1.2.2, but those will be outlined in greater

detail in the results section.

2.2. Test Organisms

Cyanobacterial cell cultures and growth media recipes were obtained from the University of Texas

(UTEX, Austin, TX) Culture Collection of Algae, the Bigelow National Center for Marine Algae

and Microbiota (NCMA, East Boothbay, ME), and the Australian National Algae Culture

Collection (ANACC, Australia). Upon receiving cultures, they were uncapped and placed in the

incubator shaker with exposure to 12 hr light/dark cycles prior to inoculation into sterile growth

media. All cultures used in this study are summarized in Table 2.

Table 2. Cultures obtained from culture collections and associated growth media type.

Species/Strain Source Media Type

Oscillatoria lutea var. contorta LB 390 UTEX Modified Bold 3N

Nodularia spumigena B 2091 UTEX BG-11

Lyngbya aestuarii LB 2515 UTEX Enriched Seawater Medium

Anabaena flos-aquae LB 2557 UTEX BG-11

Nostoc commune B1612 UTEX BG-11(-N)

Fischerella ambigua 1903 UTEX BG-11(-N)

Synechococcus elongatus L 2973 UTEX BG-11

Microcystis aeruginosa LB 2388 UTEX Bold 3N

Cylindrospermopsis raciborskii CCMP1973

Bigelow DYVm

Aphanizomenon sp. CCMP2762 Bigelow DYVm

Microcystis aeruginosa CCMP3462 Bigelow AF6

Anabaena-like sp. CCMP3421 Bigelow Black Sea

Trichodesmium ertythraeum CCMP1985 Bigelow YBCII

23

Raphidiopsis raciborskii CR3 ANCC Modified COMBO

2.2.1. UTEX

Eight cultures were obtained from UTEX and tested for their ability to grow in the incubator

chamber under an LED light in the UNLV Emerging Diseases Laboratory. These eight cultures

from UTEX were selected because they encompass different species within each genus and are

reported to produce cyanotoxins (EPA, 2019), although it was clearly stated by UTEX that none

of these cultures are toxin-producing.

2.2.1.1. Microcystis aeruginosa

After optimizing growth conditions on Microcystis aeruginosa culturing techniques at the River

Mountain Water Treatment Facility (SNWA, Henderson, NV), the first culture obtained in the

UNLV Emerging Diseases Laboratory was Microcystis aeruginosa LB 2388. UTEX recommends

growing this strain at 20°C under warm-white or cool-white fluorescent lamps in Bold 3N Media

(B3N). As noted previously, an LED light with a broader spectrum was used in place of the

recommended fluorescent lamps (Figure 6).

24

Figure 6. Full-spectrum LED light mounted in the mirrored environmental chamber used in this study.

A 15 mL aliquot of the LB 2388 strain arrived at the laboratory and 5 mL was transferred

aseptically into an Erlenmeyer flask containing approximately 200 mL of B3N media. The flask

was sealed with aluminum foil and placed in the incubator under a 12 hour light/dark cycle

provided by the LED light. Figure 7 shows Microcystis aeruginosa LB 2388 cells, but

demonstrates that visual cell counts are difficult due to the inability to distinguish individual cells,

as it is not clear if this is four cells, or two cells that have become irregularly shaped. Intracellular

inclusions can also be seen, which include carboxysomes, polyphosphate bodies, and gas-filled

vesicles for buoyancy.

25

Figure 7. 1000x photomicrograph of Microcystis aeruginosa LB 2388.

2.2.1.2. Oscillatoria lutea var. contorta

Oscillatoria lutea var. contorta UTEX LB 390 has a recommended growth temperature of 20°C,

but in Modified Bold 3N Media, which is standard B3N with two extra vitamin solutions added,

as detailed in Appendix I. Under the 100x objective lens, the name Oscillatoria becomes quite

obvious because the long filaments gently move and oscillate around independently (Figure 8).

Figure 8. 1000x magnification photomicrograph of Oscillatoria lutea var. contorta UTEX LB 390.

26

2.2.1.3. Nodularia spumigena

This Nodularia spumigena B 2091 grows optimally at 20°C in BG-11 media.

Figure 9 depicts the barrel-shaped Nodularia cells forming a filament against a backdrop of a

matrix material that has been formed in pure culture.

Figure 9. A single filament of Nodularia spumigena B 2091 at 1000x magnification.

2.2.1.4. Lyngbya aestuarii

Lyngbya aestuarii UTEX LB 2515 grows optimally at 20°C in Enriched Seawater Medium. The

individual cells within each sheath can be best seen in the top right hand corner of the image

(Figure 10).

27

Figure 10. 1000x magnification photomicrograph of Lyngbya aestuarii UTEX LB 2515. The arrow points to an individual cell within a sheath of multiple cells.

2.2.1.5. Nostoc commune

This species was isolated from soil in Austin, Texas. This Nostoc commune B 1621 culture grows

at an optimal temperature of 20°C in BG-11(-N) Media, which is standard BG-11 media without

sodium nitrate added. Figure 11 shows chains of Nostoc cells at 400x magnification.

28

Figure 11. 400x magnification photomicrograph of Nostoc commune B 1621.

In a culture flask, Nostoc forms cloudy clumps of cells, as demonstrated in Figure 11.

2.2.1.6. Fischerella ambigua

The Fischerella ambigua 1903 strain grows optimally in BG-11(-N) media at 20°C. No clear photo

is available of this culture.

2.2.1.7. Synechococcus elongatus

This Synechococcus elongatus 2973 culture optimal growth temperature is identified as 30°C,

while growing best in BG-11 Media. Synechococcus is classified as a picocyanobacterium, which

is generally known as cyanobacteria that are too small to form traditional visual accumulations of

growth that would be considered a CyanoHAB. Cyanobacteria that do not produce blooms, but

still produce cyanotoxins are a concern if water authorities base water quality evaluations on visual

identifications of blooms in drinking water reservoirs. The general interest in obtaining this culture

29

was to determine if Synechococcus has the gene that produces BMAA, since it has been reported

in the literature that it does not produce microcystin. A culture that produces BMAA but does not

produce visual algal blooms would be very interesting to investigate further as a model system. As

seen in Figure 12, Synechococcus cells are very small compared to any other culture shown

previously in this section.

Figure 12. Synechococcus elongatus 2973 cells at 1000x magnification in pure culture.

2.2.1.8. Anabaena flos-aquae

Anabaena flos-aquae 2557 grows optimally at 20°C in BG-11 media. Although several of the

previously mentioned cultures produce an extracellular matrix in pure culture, the physical

characteristics of Anabaena flos-aquae 2557 are tremendously different than any other culture

obtained from UTEX. This culture is characterized by a solid blob of thick, gelatinous seaweed-

like material.

30

2.2.2. National Center for Marine Algae and Microbiota (NCMA)

Among many different culture collections around the world, the best option for obtaining a

microcystin-producing Microcystis aeruginosa culture appeared to be from the Bigelow

Laboratory for Ocean Sciences in East Boothbay, Maine which is the site of the NCMA.

2.2.2.1. Microcystis aeruginosa

NCMA identifies Microcystis aeruginosa CCMP3462 as toxin-producing, and the website

suggests culturing AF6 or DYVm media in a temperature range of 20 to 26°C. Coincidently, after

ordering this culture from NCMA, it was later discovered that Microcystis aeruginosa Kützing is

the same strain that was identified in the 2015 microcystin-producing CyanoHAB in Lake Mead

(Beaver et al., 2018). It was observed that the NCMA recommends the use of different media (AF6

or DY-Vm) for growing M. aeruginosa than what is used by the Southern Nevada Water Authority

(B3N). Due to this discrepancy, a literature review was performed to determine the appropriate

media to be used in this study at UNLV. A study reported the growth and microcystin production

of a strain of Microcystis aeruginosa under varying nutrient conditions (Bortoli et al., 2014).

According to this study, the highest microcystin content during exponential phase was produced

by cultures grown in BG-11 media, and the lowest microcystin production during exponential

phase was seen with the use of B3N media. Therefore, BG-11 was selected as the growth media

for use in this study.

2.2.2.2. Cylindrospermopsis raciborskii

Cylindrospermopsis raciborskii CCMP1973 (Wolosz.), synonyms A9298 or JS1549, has an

optimal growth temperature of 18-22°C in a variety of different media, including DY-Vm, DY-V,

and DY-V+ND. In contrast to all of the previous cultures mentioned in this section, the

Cylindrospermopsis raciborskii CCMP1973 culture never grew in the UNLV Emerging Diseases

31

Laboratory despite utilizing the growth media and temperature range recommended by the NCMA.

No visible growth could be seen in the media, and extracted DNA was never detected.

2.2.2.3. Aphanizomenon

Aphanizomenon sp. CCMP2762, synonyms A11 and 386, is suggested that the optimal growth

temperature is 11-16°C in DY-Vm, or DY-V media. The culture was the least visually observable

of all cultures obtained thus far, aside from C. raciborskii that failed to grow to detectable levels.

Tiny particles were observed floating in the media.

2.2.2.4. Anabaena-like

Anabaena-like sp. CCMP3421, synonyms A13 and 189, is recommended to grow in Black Sea

media at 20°C. Unfortunately this culture dried out before more Black Sea media was obtained,

and therefore the culture was not available for further analysis.

2.2.2.5. Trichodesmium erythraeum

Trichodesmium erythraeum CCMP1985, synonym INS101, has an optimal growth temperature

range of 22-26°C in YBCII media. Like the C. raciborskii culture, this T. erythraeum culture never

grew in the laboratory at UNLV.

2.2.3. ANACC

Cylindrospermopsis raciborskii was obtained from the Australian National Algae Culture

Collection (ANACC). The CR3 strain acquired has been reported to produce 6,492 µg/g BMAA

in freshwater (Cox et al., 2005). This provided a unique opportunity to use this culture as a positive

control for BMAA production, but it was also an opportunity to reproduce these high BMAA

production levels.

32

2.2.3.1. Raphidiopsis raciborskii

The Cylindrospermopsis raciborskii CR3 strain was recently reclassified as Raphidiopsis

raciborskii according to the ANACC. This is not a new taxonomical concern, with a

recommendation to place both of these organisms within the same genus having been proposed

over 10 years ago (Stucken et al., 2008). The ANACC doesn’t sell media, so four media types

were purchased to determine which is more effective: Jaworski’s media (Culture Collection of

Algae and Protozoa, Scotland), DYV (Bigelow National Laboratory NCMA), BG-11 (UTEX), and

Modified Combo media (UTEX). The components of each media type are compared in Table 3.

Table 3. Media recipes used to culture R. raciborskii compared to the media recipe recommended by ANACC (MLA).

MLA Jaworski DYV BG-11 Modified COMBO

NaNO3 NaNO3 NaNO3 NaNO3 NaNO3

K2HPO4 K2HPO4 K2HPO4 K2HPO4

MgSO4•7H2O MgSO4•7H2O MgSO4•7H2O MgSO4•7H2O MgSO4•7H2O

CaCl2•2H2O CaCl2•2H2O CaCl2•2H2O CaCl2•2H2O

C6H8O7

C6H8FeNO7

Na2EDTA Na2EDTA Na2EDTA Na2EDTA Na2EDTA

NaHCO3 NaHCO3 NaHCO3 NaHCO3

H3BO3 H3BO3 H3BO3 H3BO3 H3BO3

MnCl2•4H2O MnCl2•4H2O MnCl2•4H2O MnCl2•4H2O MnCl2•4H2O

ZnSO4•7H2O ZnSO4•7H2O ZnSO4•7H2O ZnSO4•7H2O

Na2MoO4 Na2MoO4 Na2MoO4 Na2MoO4

CuSO4•5H2O CuSO4•5H2O CuSO4•5H2O

Co(NO3)2•6H2O

Na2SO3 Na2S2O3·5H2O

Na2SiO3•9H2O Na2SiO3•9H2O

33

KCl KCl

FeCl3•6H2O FeCl3•6H2O FeCl3•6H2O

CoCl2•6H2O CoCl2•6H2O CoCl2•6H2O

H2SeO3 H2SeO3 H2SeO3

Na3VO4 Na3VO4

Vitamin B12 Vitamin B12 Vitamin B12 Vitamin B12

Biotin Biotin Biotin Biotin

Thiamine Thiamine Thiamine Thiamine

CR1 Soil

NH4Cl

C3H7Na2O6P

Ca(NO3)2•4H2O

EDTAFeNa

Na2HPO4.12H2O

(NH4)6Mo7O24.4H2O

2.2.4. OD600 Spectrophotometry

Following an appropriate incubation period, a volume of 2 mL of each culture was transferred to

a polystyrene spectrophotometry cuvette for analysis of optical density at 600 nm on a Spectronic

Genesys 10 Bio (Thermo Electron Corp., Madison, WI) spectrophotometer. A cuvette containing

either growth media or deionized water was used as a blank. When handling cuvettes, gloves were

used at all times to avoid smudging the sides of the cuvette associated with optics. After the blank

was established, samples were measured, and the OD600 was recorded with five separate

measurements to establish an average. It was observed that some of the solids settle very rapidly

to the bottom of the cuvette, and in this case a pipette was used to manually mix the contents of

the cuvette by drawing in a volume and dispensing it immediately a few times prior to taking a

sample reading.

34

2.2.5. Cell Counting

The total concentration of the M. aeruginosa cell suspension was determined using a Multisizer™ 3

electronic particle counter (Beckman Coulter, Inc., Miami, FL). An aliquot of the cell suspension

was diluted in filtered Isoton II solution (Beckman Coulter, Inc., Miami, FL) and enumerated using

the electronic particle counter. The particles corresponding to the cell peak were counted, and the data

were automatically adjusted for coincidence correction by the instrument. Aliquots of the enumerated

cell suspension were stored at 4C overnight for OD600 sampling and DNA extraction.

2.2.6. Disposal of Toxic Wastes

Proper disposal of cyanotoxins is essential given the fact that they are not inactivated by

autoclaving. It is for this primary reason that the decision was made in consultation with the

UNLV Institutional Biosafety Committee (IBC) and the Department of Risk Management and

Safety that all cyanobacteria waste should be incinerated (Rao et al., 2002). After a culture was

poured into a waste container containing bleach for storage until pickup for incineration, the

glass flask used in the culturing experiment often had residue on the sides of the flask from

cyanobacterial biomass. The flasks were filled with 10% bleach and allowed to sit for a

minimum of one hour. Although bleach is commonly used to disinfect liquids, there is a lack of

understanding of the survivability of spore-like cells such as akinetes during bleach contact. It is

reasonable to assume that bleach is not effective in killing all akinetes, so for this reason the

bleach contact time was often performed for several hours, and in some cases overnight if

glassware had visible biomass on the inside after emptying the liquid into the waste container.

35

2.3. TaqMan Primers and Probes

The following real-time PCR primers and probes were identified from the literature (Operon

Biotechnologies, Huntsville, AL; Applied Biosystems, Foster City, CA).

2.3.1. Universal Cyanobacteria

CYAN 108F 5’-ACGGGTGAGTAACRCGTRA-3’ (Urbach et al., 1992)

CYAN 377R 5’-CCATGGCGGAAAATTCCCC-3’ (Nubel et al., 1997)

CYAN 328R (Taq) 5’-FAM-CTCAGTCCCAGTGTGGCTGNTC-BHQ-1-3’ (Rinta-Kanto et al., 2005)

2.3.2. Microcystis-specific 16S rRNA gene

MICR 184F 5’-GCCGCRAGGTGAAAMCTAA-3’ (Neilan et al., 1997)

MICR 431R 5’-AATCCAAARACCTTCCTCCC-3’ (Neilan et al., 1997)

MICR 228 F (Taq) 5’-FAM-AAGAGCTTGCGTCTGATTAGCTAGT-BHQ-1-3’ (Rinta-Kanto et al., 2005)

2.3.3. Phycocyanin

Phycocyanin is a photosynthetic pigment produced only by cyanobacteria. The use of this

primer/probe is intended to assess if targeting phycocyanin genes is a better approach than

targeting universal 16S rRNA genes.

188F PC 5’-GCTACTTCGACCGCGCC-3’ (Kurmayer et al., 2003)

254R PC 5’-TCCTACGGTTTAATTGAGACTAGCC-3’ (Kurmayer et al., 2003)

PC (Taq) 5’-FAM-CCGCTGCTGTCGCCTAGTCCCTG-TAMRA-3’ (Kurmayer et al., 2003)

2.3.4. Microcystin toxin gene cluster

The Microcystin toxin biosynthetic gene cluster consists of genes encoding peptide synthetase,

polyketide synthase, or modifying enzymes and mapped to a 55 kb cluster. Genes in the 55 kb

36

cluster include mcyA through mcyJ. The following three primer and probe sets were chosen to

determine if there is any difference between these genes as targets in TaqMan QPCR. The gene

cluster is not limited to Microcystis species, as it is also well described in Nostoc, Anabaena,

Nodularia, Fischerella, and Planktothrix, (Heck et al., 2017) among others.

2.3.4.1. mcyB (Kurmayer et al., 2003)

mcyB 30F 5’-CCTACCGAGCGCTTGGG-3’

mcyB 108R 5’-GAAAATCCCCTAAAGATTCCTGAGT-3’

mcyB P (Taq) 5’-FAM-CACCAAAGAAACACCCGAATCTGAGAGG-TAMRA-3’

2.3.4.2. mcyD (Kaebernick et al., 2000, Rinta-Kanto et al., 2005)

mcyD F2 5’-GGTTCGCCTGGTCAAAGTAA-3’

mcyD R2 5’-CCTCGCTAAAGAAGGGTTGA-3’

mcyD F2 (Taq) 5’-FAM-ATGCTCTAATGCAGCAACGGCAAA-BHQ-1-3’

2.3.4.3. mcyE (Sipari et al., 2010)

mcyE F 5’-AAGCAAACTGCTCCCGGTATC-3’

mcyE R 5’- CAATGGGAGCATAACGAGTCAA-3’

mycE P (Taq) 5’- FAM-CAATGGTTATCGAATTGACCCCGGAGAAAT-TAMRA-3’ 2.3.5. Cylindrospermopsin toxin gene cluster (Campo et al., 2013)

The cylindrospermopsin toxin gene cluster consists of genes cyrA through cyrO. The target of this

primer/probe set is the gene cyrJ.

cyrJ207F 5’- CCCCTACAACCTGACAAAGCTT-3’

cyrJ207R 5’- CCCGCCTGTCATAGATGCA-3’

cyrJ207P (Taq) 5’- FAM-AGCATTCTCCGCGGATCGTTCAGC-TAMRA-3’

37

2.3.6. Anabaena-specific 16S rRNA gene (Sipari et al., 2010)

Ana611F 5’- CTAGAGTAGTCACTCACGTC-3’

Ana737R 5’- GGTTCTTGATAGTTAGATTGAGC-3’

Ana672P (Taq) 5’- FAM-CAAGTTCCCACAATTCTTGGATTAGCAGC-TAMRA-3’

2.3.7. Synechococcus-specific RuBisCO gene (Wawrik et al., 2009)

Syn rbcL F 5’- CATCAAGCTGTCCGAG-3’

Syn rbcL R 5’- TGTTGGCYGTGAAGCC-3’

Syn rbcL P (Taq) 5’- FAM-TCACTACCTCAACGTGACCGC-TAMRA-3’

2.3.8. Primer/Probe Working Stocks

It was necessary to re-suspend each primer in TE buffer (Teknova, Hollister, CA) to obtain 100

µM primer stocks. All primer and probe re-suspensions were performed in the PCR clean room

using the CleanSpot PCR/UV workstation (Coy Laboratory Products, Grass Lake, MI). Primers

were resuspended in TE buffer to achieve 100 µM and 10 µM freezer and working stocks,

respectfully, aliquoted, and frozen at -70°C. Probe stocks (100 µM) were diluted with TE to form

10 µM working stocks, aliquoted, and frozen at -70°C.

2.3.10. Lake Water

Samples of Colorado River water were provided by the Southern Nevada Water Authority for use

in this study. The samples are free of any oxidants or treatments for quagga mussel control.

Samples were stored in sterile bottles at 4°C in the dark prior to use.

2.4. DNA Extraction

2.4.1. Amicon Ultra Filtration

38

DNA was extracted according to the Amicon size-exclusion DNA extraction protocol (EMD

Millipore, Chicago, IL). A volume of 250 µL of a cyanobacterial culture was added to 2 mL bead

beater tube containing 0.25 g of sterile acid-washed 0.1 mm and 0.5 mm zirconia/silica glass beads.

A volume of 250 µL 100mM NaPO4/EDTA/Tween-20 (Teknova, Hollister, CA) was then added

to a bead-beater tube and immediately agitated at 5000 rpm for 180 sec (3 min) in a mini-bead

beater (Biospec Products, Bartlesville, OK). Following bead-beating samples were cooled for ~ 2

minutes in the refrigerator at 4°C prior to quick-spin centrifugation to 5200x g. Without disturbing

the glass bead pellet, ~400 µL of supernatant was transferred to an Amicon filter that had already

been seated in an Amicon Ultra 0.5 mL vial using sterile forceps. The filter/vial combo were then

centrifuged at 5200x g for 3 min. Using sterile forceps, the Amicon filter was then transferred to a

new 0.5 mL Amicon Ultra vial for the first of four washing steps. In Wash 1, 400 µL of TE buffer

(Teknova, Hollister, CA) was added directly to the Amicon filter, and the filter/vial combo was

centrifuged at 5200x g for 3 min. After transferring the Amicon filter to a new Amicon Ultra vial,

400 µL of TE buffer were added to the Amicon filter for Wash 2, and again spun at 5200x g for 3

min. For Wash 3, the filter was again moved to a new Amicon Ultra vial, with 400 µL of TE buffer

added prior to centrifugation at 5200x g. In the final wash step, 400 µL of HyPure Molecular

Biology Grade Water (HyClone Laboratories, San Angelo, TX) were added to the Amicon filter,

and the filter/vial combo were spun in 30 second increments until the filter volume was

approximately 100 µL. This final eluate was transferred to a 1.5 mL microcentrifuge tube, heated

to 65°C for 2 min, cooled at 4°C for 2 mins, and stored at -70°C.

39

2.4.1.1. DNA Extraction Optimization

NaPO4 is used as a buffer during bead beating because DNA extraction is a pH-sensitive process.

Three different combinations of culture and buffer totaling 500 µL was used to assess the

combination that produces the highest DNA yield/lowest Ct.

Scenario 1: 500 µL culture, 0 µL NaPO4 buffer



Following DNA extraction using an M. aeruginosa pure culture, the primers and probes used in

this assessment were Universal Bacterial, consisting of the forward primer NadF 5’-

TCCTACGGGAGGCAGCAGT-3’, the reverse primer NadR 5’-

GGACTACCAGGGTATCTAATCCTGTT-3’, and the universal probe UnivP 5’-FAM-

CGTATTACCGCGGCTGCTGGCAC-TAMRA-3’ (Nadkarni et al., 2002).

2.4.2. Freeze-Thaw DNA Extraction Method

Sample volumes of approximately 250 µL were aseptically transferred to a 2 mL microcentrifuge

tube. The tube was placed into a freezer at -17°C for 10 min, and then transferred into a 65°C water

bath for 2 min. The tube was then transferred back to the -17°C freezer for 10 mins, followed by

rapid vortexing for approximately 1 min. The cells were then spun down at 5200x g for 1 min, and

150 µL of supernatant was transferred to a 1.5 mL microcentrifuge tube and stored at -70°C.

2.5. Quantitative Polymerase Chain Reaction (QPCR)

After establishing the plan for the assay and labeling the 1.5 mL MasterMix tube and one 0.6 mL

tube per sample, the reagents that were frozen at -70°C, including molecular-grade water, primers,

40

probe, and DNA samples were removed from the freezer and allowed to thaw at room temperature

in sterilized microcentrifuge racks. The TaqMan Fast Advanced Master Mix (Applied Biosystems,

Foster City, CA) Mastermix was not removed from the 4°C cooler until immediately before use.

While frozen reagents and samples were thawing, the 96-well plate plan was entered into the

Sequence Detection System (SDS) software according to the UNLV Emerging Diseases

Laboratory SOP. Sub-mastermixes were then prepared in the clean room inside a bleached

CleanSpot PCR/UV Work Station (Coy Lab Products) previously disinfected with 20 mins UV

light exposure. The volumes of molecular-grade water, 2x Fast Universal MasterMix, 10 µM

forward primer, 10 µM reverse primer, and 10 µM probe were added to the 1.5 mL MasterMix

tube and vortexed. No DNA is introduced to the CleanSpot PCR/UV Work Station throughout the

duration of MasterMix preparation. Moving the 0.6 mL MasterMix tubes to the AirClean 600 PCR

Workstation, 11 µL of vortexed DNA sample from each thawed tube is added to each 0.6 mL tube.

After vortexing each MasterMix/DNA combined sample, 25 µL was added to each well on the

MicroAmp Fast Optical 96-well plate, with each sample, including positive and negative controls,

in duplicate on the plate. Once all duplicate samples were added to the plate, a sheet of MicroAmp

Optical Film was added to cover the plate and carefully sealed and placed into the 7900HT Fast

Real-Time PCR System (Applied Biosystems, Foster City, CA). Fast mode cycling conditions

consisted of initial 50°C incubation for 2 min and denaturation at 95°C for 20 sec, followed by 40

cycles of 95°C denaturation for 1 sec and elongation at 60°C for 20 sec. The run is then started,

and amplification curves are tracked in real-time. After completion of the run, the data were

analyzed according to the SDS SOP.

41

2.5.1. Internal Positive Control (IPC)

TaqMan Exogenous Internal Positive Control (IPC) Reagents (Applied Biosystems, Foster City,

CA) were used to determine if inhibitors were present in the PCR reaction. The kit as ordered from

ThermoFisher contains the following reagents: 10X Exo IPC Mix, 10X Exo IPC Block, and 50X

Exo IPC DNA. A feature of the kit includes pre-designed primers and a TaqMan probe, which

eliminates the need to design this assay. The IPC probe is 5’-labeled with VIC, allowing the IPC

reaction to take place in the same well with FAM-labeled probes designed for a target sequence.

The IPC reaction performs optimally using the TaqMan Universal PCR Master Mix (Applied

Biosystems, Foster City, CA). The sequences of the pre-designed primers and probe in this kit are

proprietary.

Although negative controls are a standard feature of any quality-controlled PCR reaction, it is

essential to apply quality control to false-negatives as well. Using the reaction preparation and

thermocycling conditions described previously in Section 2.5, the IPC assay helps to determine

the difference between negative reactions due to a lack of a target sequence, negative reactions due

to the presence of an inhibitor (i.e., false negative), as well as partial inhibition.

2.5.2. QPCR Standard Curve

After performing a cell count according to Section 2.2.5, the 2 mL of the same culture was used to

determine the OD600 optical density corresponding to that cell count, using BG-11 media as a

blank. Two 1:10 dilutions were prepared from the culture and then evaluated for OD600. DNA was

extracted from all three samples used in OD600 measurements, and 40-cycle TaqMan QPCR was

performed to determine Ct values corresponding to each OD600/cell count, providing a standard

curve relating Ct and cell concentration.

42

2.6. Toxin Measurement

Toxins were analyzed at the River Mountain Water Treatment Facility as described below.

2.6.1. Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS)

Analytes were purchased from Enzo Life Sciences (Farmingdale, NY) or Abraxis BioScience (Los

Angeles, CA) as neat material or in solution. Isotopically labeled analytes were purchased from

Abraxis or Cambridge Isotope Laboratories (Xenia, OH) as solutions. Neat material was dissolved

in 1 mL of methanol and all stocks were stored at -20°C. Using individual stock solutions, infusion

tests were performed to develop and optimize instrument parameters for all analytes using both

electrospray (ESI) positive and negative ionization modes on a SCIEX 4000 QTRAP liquid

chromatography/tandem mass spectrometry (LC/MS/MS) system (Ontario, Canada). A working

stock mix was made for the unlabeled analytes with a final concentration of 500 µg/L and used to

finalize source parameters and optimize the LC program. LC separation was achieved on a

Phenomenex Luna C18, 5 μm column, 150 x 4.6 mm (Torrance, CA). The mobile phase consisted

of a binary gradient using 0.1% formic acid in deionized water and 0.1% formic acid in acetonitrile.

Injection volume was 50 μL. All analytes were included in both ESI positive and negative methods,

except for anatoxin-a, which only ionizes in the ESI positive mode. After method optimization, a

working stock mix of the isotopically labeled analytes was prepared at 250 µg/L. Using both

working stock mixes, a calibration curve was made that ranged from 0.25 to 50 µg/L. The IS

concentration in these samples was 5 µg/L. Final ESI positive and negative methods were created

using scheduled MRM.

Quantitation methods were created using isotope dilution for all analytes, except as noted below.

Analytes with no available isotopically labeled analogues were linked to an isotope used in the

43

method that was the closest structurally and/or closest in retention time. Instrument detection limits

(IDLs) were performed for both ESI positive and negative methods.

2.6.2 Enzyme-Linked Immunosorbent Assay (ELISA)

An Abraxis ELISA microtiter plate (Eurofins Technologies, Hungary) with antibody-coated wells

was used to determine BMAA concentrations in samples. Lyophilized BMAA standards were

provided in the ELISA kit, in the range of 0, 5, 25, 100, 250, and 500 ng/mL (ppb). These standards

are subsequently referred to as Std 0, Std 1, Std 2, Std 3, Std 4, and Std 5. Standards were

reconstituted by adding 1 mL DI water to each standard vial and vortexed. The 5x concentrated

wash buffer was diluted 1:5 by adding 400 mL of DI water to the 100 mL bottle. Microtiter plate

columns are labeled 1 through 12, and rows are labeled A through H, resulting in a total of 96

wells (12 x 8). The plate and reagents were adjusted to room temperature prior to beginning the

assay. A volume of 100 µL of each standard was loaded in duplicate wells, with Std 0 loaded in

wells A1 and B1, Std 1 in wells C1 and D1, Std 2 in wells E1 and F1, Std 3 in wells G1 and H1,

Std 4 in wells A2 and B2, and Std 5 in wells C2 and D2. Then 100µL of each environmental

sample was loaded in subsequent wells. Using a multi-channel pipette, 50 µL of BMAA-HRP

conjugate solution was added to each well, followed by 50 µL of rabbit anti-BMAA antibody

solution added to each well. All wells were then covered with parafilm and mixed in a circular

motion for 30 sec, followed by incubation at room temperature for 90 mins. The parafilm cover

was then removed and each well was washed with 250 µL of 1x wash buffer, and then each step

was repeated for a combined total of 4 washes. The wells were then air-dried through inversion,

and 150 µL of TMB substrate color solution was added to each well, parafilm was again applied,

the plate was mixed in a circular motion for 30 seconds, and the plate was allowed to incubate at

room temperature for 30 mins in the absence of direct sunlight. Following incubation, 100 µL of

44

stop solution was then applied to each well in the same sequence that the TMB substrate color

solution was added. Within 15 mins of the addition of the stop solution, absorbance at 450 nm was

read using a microplate ELISA photometer. A standard curve was constructed by plotting %B/B0

on the y-axis, and log BMAA concentration on the x-axis. The %B/B0 ratio is found by dividing

the mean absorbance value of the Std 1-5 duplicates by the mean of the Std 0 duplicates. The

detection limits are anything lower than Std 1 (5 ppb) or higher than Std 5 (500 ppb).

Environmental sample BMAA concentrations are interpolated in ppb (or ng/mL) using the

standard curve shown in Figure 13.

Figure 13. Example standard curve used to determine BMAA concentrations of environmental samples from absorbance data (Source: Southern Nevada Water Authority, unpublished).

y = -1.1664x + 3.1837R² = 0.9817

0

0.5

1

1.5

2

2.5

3

0 0.5 1 1.5 2 2.5

Me

an A

bso

rban

ce a

t 4

50

nm

log(X) BMAA Concentration (ppb)

BMAA Standard Curve

Linear (BMAA Standard Curve)

45

2.6.3. Seafood sampling

Seafood was purchased at a Las Vegas grocery store. Each of the seafood products were aseptically

removed from the packaging and placed into a sample vial. The mass of the seafood introduced to

the vials was not measured. RO water was also introduced to the sample vial, sealed, and then

shaken vigorously by hand to physically digest the material.

2.7. Sample Protocol for Rapid Detection of Microcystin-Producing Genes

1. Obtain a sample

There were no blooms available to sample at Lake Mead during this study, but simulated algal

blooms produced at UNLV provided the opportunity to develop this protocol. Figure 14 below

shows buoyant Microcystis aeruginosa (Kützing) at the surface of a mixture of autoclaved Lake

Mead water and BG-11 growth media that was originally grown in a 400 mL total volume (200

mL BG-11 + 200 mL autoclaved lake water + 5 mL M. aeruginosa pure culture), and later

aliquoted into this smaller ~200 mL volume for development of surface accumulation.

46

Figure 14. Buoyant M. aeruginosa (Kützing) cells accumulated at the water surface.

At Lake Mead, a grab sample ideally using a sterile spatula to transfer growth to a 50 mL Falcon

tube should be taken of the surface accumulation, with careful attention not to allow the growth

to contact the skin of the person taking the sample. In windy conditions, respiratory protection

such as an N-95 mask should be worn to avoid inhaling aerosolized droplets.

2. Transport the sample to the laboratory

On wet ice, the grab sample is transported to the laboratory.

3. Perform freeze-thaw protocol to lyse cells (Total time = 22 mins)

Transfer 250 µL of sample from the surface to a sterile 1.5 mL microcentrifuge tube. The sample

is then placed into a freezer at -17°C for 10 minutes. The tube is then transferred to a water bath

47

at approximately 65°C using a floating microcentrifuge rack. After two minutes, the sample tube

is then returned to the -17°C freezer for 10 minutes.

4. Obtain a DNA sample (Total time = 3 mins)

Following freez/thaw, the 250 µL sample is rapidly vortexed for one minute, followed by

centrifugation at 5200xg for one minute to pellet cellular material. A pipette is used to carefully

remove any surface scum that did not become pelleted, and a new pipette tip is used to carefully

remove 100 µL of volume from the supernatant without disturbing the cell pellet. This crude

DNA sample is then stored at 4°C.

5. Preparation of the PCR MasterMix (5 mins, performed during Step 3)

While samples are being freeze-thawed, the PCR MasterMix can be made and aliquoted as

indicated previously. From the MasterMix tube, sub-MasterMixes are made in labeled 0.6 mL

tubes at a volume of 44 µL each. The total time to do this for three samples (environmental

sample, positive control, and negative control) is 5 minutes, and does not add to the overall time

associated with the assay due to being performed while the environmental sample is in the

freezer. Figure 15 shows the clean work station used in this example.

48

Figure 15. MasterMix preparation in a clean work station.

6. DNA added to sub-MasterMix (Total time = 4 mins)

The environmental sample, positive control (M. aeruginosa Kützing 10x concentrated DNA),

and negative control (PCR-grade water) sub-MasterMix tubes are then transferred to a dedicated

work station to add 11 µL of DNA template to each tube in a step that takes 4 minutes for 3

different tubes.

7. Sub-MasterMix/DNA solution added to the 96-well plate (Total time = 6 mins)

According to the plan established on the TaqMan Assay 7900 Fast Mode PCR, 25 µL of each

PCR reaction is added to each well, which in this example is wells A1 and A2 for the PCR-grade

water negative control, wells C1 and C2 for the environmental sample, and wells E11 and E12

for the Microcystis positive control.

49

8. TaqMan QPCR cycle (Total time = 39 mins)

The 96-well plate is loaded into the 7900HT unit as shown in Figure 16.

Figure 16. The 96-well plate prepped for loading into the 7900HT.

The 40-cycle thermocycler reaction is then started, with visual tracking of the amplification

curves during the 39 minute process.

In a total time of 74 minutes (1 hour, 14 minutes), a sample collected from an algal bloom or

wind-driven surface accumulation that is brought to the laboratory can be assayed by one

technician to determine if it is positive or negative for microcystin-production genes.

2.8.1. Protocol Modification in the Absence of a Visible Bloom

The 8-step protocol described above is a rapid and cost-efficient method of identifying

Microcystis from surface blooms or wind-driven aggregations of cells, but a slight adjustment in

50

the protocol involving concentration of the sample by centrifugation is necessary to detect

Microcystis in samples without visible cell accumulation. The modified protocol for detecting

low-abundance Microcystis involves changes to Step 3, the freeze-thaw protocol, and the

sampling protocol, with the use of a submerged sampling bottle. First, the cultures were shaken

by hand to mimic wind-driven surface water mixing without surface aggregation. A volume of 4

mL from each flask was transferred to a 15 mL Falcon tube, and 2 mL microcentrifuge tubes

were labeled MC+ (Microcystis present) and MC- (Microcystis absent). A volume of 1.25 mL

was transferred from the Falcon tube to each of the corresponding microcentrifuge tubes and

then centrifuged at 5200xg for one minute. The supernatant was removed by pipette, with careful

attention to avoid disturbing the cell pellet. Another 1.25 mL was then added to each

microcentrifuge tube, and the tubes were again centrifuged at 5200xg for one minute. The

remaining supernatant was removed without disturbing the cell pellet, and then the pellet was re-

suspended in 250 µL HyPure molecular grade water.

At this point, the protocol becomes the same as previously described, with these 250 µL 10x

concentrated samples placed into the freezer at -17°C for 10 minutes in preparation for transfer

to the 65°C water bath.

51

Chapter 3. Data and Results

3.1. Simulated Algal Blooms

Objective 1: Establish a surrogate (simulation) system to grow M. aeruginosa in Lake Mead

conditions.

The following growth conditions were established in cultures that were grown in the same

environmental chamber, under the same LED light on 12 hour light/dark cycles, and at the same

temperatures:

1. M. aeruginosa pure culture growth curve in BG-11 media (see Appendix V for media

components)

2. M. aeruginosa growth in autoclaved lake water amended with BG-11 media

3. M. aeruginosa growth in lake water amended with BG-11 media

4. M. aeruginosa growth in lake water amended with 1/5 diluted BG-11 media

5. Lake water growth in BG-11 and Modified Combo growth media

6. Lake Water controls with no growth media

3.1.1. M. aeruginosa growth curve in BG-11 media

In accordance with a protocol from cyanobacterial culturing techniques used at the River

Mountain Water Treatment Facility in Las Vegas (Wert et al., 2013), the standard method of

establishing pure cultures is to aseptically inoculate a 5 mL sample from a pure culture into 200

mL of growth media. In this case, the growth medium used is BG-11 (UTEX). Four separate

cultures were sampled weekly or bi-weekly to obtain the following four growth curves (Fig 17):

52

Figure 17. 30-day growth curves associated with standard pure culture replicates. Cultures 1 through 4 are all 200 mL BG-11 + 5 mL Microcystis pure cultures.

Each of the time-zero OD600 measurements has a non-zero cell density reading that is associated

with the amount of cells that were spiked into the media from a pure culture. Cultures are

numbered 1 through 4 by the date they were set up, with Culture #1 inoculated on 8/18/2019,

Culture #2 inoculated on 10/15/2019, Culture #3 inoculated on 11/8/2019, and Culture #4

inoculated on 1/9/2020.

3.1.2. M. aeruginosa growth curve in autoclaved lake water amended with BG-11 media

To determine if the lake water has characteristics or substrates that influence the growth of

Microcystis, cultures containing 200 mL BG-11 media mixed with 200 mL lake water were

0.001

0.01

0.1

1

0 5 10 15 20 25 30

OD

60

0

Time (days)

200 mL BG-11 + 5 mL Microcystis aeruginosa Kützing

Bottle Set #1

Bottle Set #2

Bottle Set #3

Bottle Set #4

53

inoculated with 5 mL of M. aeruginosa culture aliquots. Figure 18 displays the 30-day growth

curves associated with this experiment performed in triplicate.

Figure 18. 30-day growth curves associated with standard pure culture replicates. Cultures 1 through 4 are all 200 mL BG-11 + 200 mL autoclaved lake water + 5 mL Microcystis pure cultures.

Culture #1 was inoculated on 8/29/2019, Culture #2 was inoculated on 10/22/2019, and Culture

#3 was inoculated on 11/8/2019. Although the total volumes differed between this assay (405

mL total) and the pure culture described in Section 3.1.1. (205 mL), the growth kinetics were

similar.

3.1.3. M. aeruginosa growth curve in lake water amended with BG-11 media

0.001

0.01

0.1

1

0 5 10 15 20 25 30 35

OD

60

0

Time (days)

200 mL BG-11 + 200 mL Autoclaved Lake Water + 5 mL Microcystis aeruginosa Kützing

Bottle Set #1

Bottle Set #2

Bottle Set #3

54

A culture consisting of 200 mL BG-11 media, 200 mL lake water (not autoclaved), and 5 mL M.

aeruginosa pure culture was inoculated on 8/18/2019, and is shown in Fig 19 as Culture

200+200.

Figure 19. Cultures mimicking a CyanoHAB in Lake Mead.

Compared to the cultures described in Sections 3.1.1. and 3.1.2, this was the first culture that did

not display exponential growth in the first 10 days and reaching OD600 of 0.2 to 0.3 in 30 days.

The total volume of 405 mL used in Section 3.1.2. cultures had already grown similarly to the

205 mL pure culture in Section 3.1.1, so a second culture shown in Figure 39 consisting of 100

mL BG-11 + 100 mL lake water (not autoclaved) + 5 mL M. aeruginosa inoculum was prepared

on 11/8/2019 to evaluate growth in 205 mL total volume with non-sterile lake water. This

0.001

0.01

0.1

1

0 5 10 15 20 25 30 35

OD

60

0

Time (days)

BG-11 + Lake Water + Microcystis aeruginosa Kützing

200+200

100+100

55

(100+100) culture resulted in OD600 measurements that were similar to growth observed in 3.1.1.

and 3.1.2.

3.1.4. M. aeruginosa growth curve in lake water amended with 80% less BG-11 media

To assess the growth kinetics on M. aeruginosa in lake water with less available nutrients, a

culture was prepared on 8/19/2019 with 40 mL BG-11, 200 mL lake water, and 5 mL M.

aeruginosa inoculum. Figure 19 demonstrates a curve with the presence of a 10-day lag phase

that was not seen in previous cultures, but reaches an OD600 value of 0.3 by day 30 of incubation,

which is similar to the results in Sections 3.1.1-3.1.3.

Figure 20. Culture with media reduced to 40 mL BG-11.

0.01

0.1

1

0 5 10 15 20 25 30 35 40

OD

60

0

Time (days)

40 mL BG-11 + 200 mL Lake Water + 5 mL M. aeruginosa

Bottle Set #1

56

3.1.5. Lake water growth curve in BG-11 growth media

A culture was prepared without inoculating M. aeruginosa in order to determine if M.

aeruginosa is naturally present in Lake Mead, and if microbes present in the lake water will

grow in BG-11 growth media, as shown in Figure 20.

Figure 21. Lake Water + BG-11 media growth curve.

3.1.6. Lake Water Controls

As a control, lake water was added to a sterile flask and incubated in the environmental chamber

for 30+ days to determine if exposure of lake water to the LED light will promote growth. OD600

measurements were taken periodically, and no absorbance at OD600 was ever detected. As

additional controls, in the event the lake water did grow something in the absence of growth

0.010

0.100

1.000

0 10 20 30 40 50

OD

60

0

Time (days)

50mL Lake Water + 50mL BG-11

50mL lake H2O + 50mL BG-11

57

media, two additional controls were performed. First, a culture consisting of only autoclaved lake

water was incubated for 30+ days, and no visual change was detected in the flask. Also, a culture

consisting of autoclaved lake water + BG-11 media was also incubated for 30+ days, with no

visual growth observed. Nothing was expected to grow in these controls, so the purpose served

to demonstrate that the growth media was not contaminated.

3.2. TaqMan QPCR

Objective 2. Use QPCR to quantify the density of M. aeruginosa from the surrogate system.

3.2.1. Universal Cyanobacterial Primers and Probes

The specificity of universal cyanobacterial primers and probes was assessed after all of the

cultures obtained from collections had been cultivated in the laboratory (Table 4). A volume of

250 µL was used for DNA extractions, elution volume was 100 µL, and 5 µL of template was

used for PCR.

Table 4. Detection of diverse cyanobacteria using universal cyanobacterial primers and probes (CYANO). SE – S. elongatus; FA – F. ambigua; NS – N. spumigena; LA – L. aesuarii; AF – A. flos-aquae; OL – O. lutea; NC – N. commune; C. diff – C. difficile (Negative control); NTC – No Template Control. Each abbreviated species name is also followed by either the number 0, -1, or -2. This system indicates if the DNA extract used was undiluted (100), diluted 1/10 (10-1), or diluted 1/100 (10-2). Ct = cycle at which fluorescence (amplicon) is 1st detected.

Sample Name

Detector Name

Reporter Ct

NTC CYANO FAM Undetermined


SE0 CYANO FAM 15.6

SE0 CYANO FAM 15.6

SE-1 CYANO FAM 19.6

SE-1 CYANO FAM 19.4

FA0 CYANO FAM 17.1

FA0 CYANO FAM 17.3

58

FA-1 CYANO FAM 21.2

FA-1 CYANO FAM 21.3

NS0 CYANO FAM 15.2

NS0 CYANO FAM 15.1

NS-1 CYANO FAM 18.9

NS-1 CYANO FAM 18.9

LA0 CYANO FAM 20.4

LA0 CYANO FAM 20.4

LA-1 CYANO FAM 24.5

LA-1 CYANO FAM 24.6

AF0 CYANO FAM 18.0

AF0 CYANO FAM 17.9

AF-1 CYANO FAM 21.9

AF-1 CYANO FAM 22.0

OL0 CYANO FAM 18.8

OL0 CYANO FAM 18.9

OL-1 CYANO FAM 19.6

OL-1 CYANO FAM 19.6

NC0 CYANO FAM 16.7

NC0 CYANO FAM 16.6

NC-1 CYANO FAM 20.5

NC-1 CYANO FAM 20.6

C diff -2 CYANO FAM Undetermined

C diff -2 CYANO FAM 39.0903

All other DNA extracts were amplified with undiluted Cts of 20 or less. It is also noted that the

average Ct difference between undiluted and diluted DNA extracts seems consistent across all

species, approximately 4 Cts. The C. difficile 10-2 bacterial negative control was expected to

have an undetermined Ct, but one of the replicates resulted in Ct 39. This is assumed to be a

false-positive because the same DNA sample produces Ct 21 using universal bacterial primers

and probes.

59

New cultures were obtained from the Bigelow National Laboratory, and another assessment

using universal cyanobacterial primers and probes was performed using only the diluted samples.

This procedure kept the 100 DNA stocks for a longer time due to the use of 10-1 working stocks.

Table 5 shows the results of a PCR run including DNA extracted from new cultures.

Table 5. Detection of new cultures using universal cyanobacterial primers and probes. All DNA extracts were diluted 1/10, except the C. difficile DNA was diluted 1/1000. ANT – Anabaena toxic; APH – Aphanizomenon; CR – Cylindrospermopsis raciborskii; MAT – Microcystis aeruginosa toxic; S – Synechococcus elongatus new culture; TRI – Trichodesmium erythraeum.

Sample Name

Detector Name

Reporter Ct Previous Ct

NTC CYANO FAM Undetermined Undetermined

NTC CYANO FAM Undetermined Undetermined

SE-1 CYANO FAM 19.7 19.6

SE-1 CYANO FAM 19.8 19.4

FA-1 CYANO FAM 21.4 21.2

FA-1 CYANO FAM 21.5 21.3

NS-1 CYANO FAM 19.7 18.9

NS-1 CYANO FAM 19.6 18.9

LA-1 CYANO FAM 24.5 24.5

LA-1 CYANO FAM 24.6 24.6

AF-1 CYANO FAM 22.1 21.9

AF-1 CYANO FAM 22.2 22.0

OL-1 CYANO FAM 19.6 19.6

OL-1 CYANO FAM 19.7 19.6

NC-1 CYANO FAM 20.4 20.5

NC-1 CYANO FAM 20.4 20.6

MAT-1 CYANO FAM 31.2 N/A

MAT-1 CYANO FAM 31.1 N/A

CR-1 CYANO FAM Undetermined N/A

CR-1 CYANO FAM Undetermined N/A

ANT-1 CYANO FAM 37.1 N/A

ANT-1 CYANO FAM 38.8 N/A

S-1 CYANO FAM 20.5 N/A

60

S-1 CYANO FAM 20.5 N/A

APH-1 CYANO FAM 33.9 N/A

APH-1 CYANO FAM 34.0 N/A

TRI-1 CYANO FAM Undetermined N/A

TRI-1 CYANO FAM Undetermined N/A

Cdiff-3 CYANO FAM Undetermined N/A

Cdiff-3 CYANO FAM Undetermined N/A

No growth had been visible to the naked eye in the Cylindrospermopsis raciborskii and

Trichodesmium erythraeum cultures, so the absence of Ct values in those samples does not

suggest a lack of specificity of the universal cyanobacterial primers and probes. The Cts match

well with the Cts from the previous PCR run, indicating good repeatability for replicating PCR

runs performed on different days on the same well-mixed DNA samples. All 11 cultures that

grew in the lab were detectable using universal cyanobacterial primers and probes.

3.2.2. Assessing Inhibition

Cyanobacteria produce a wide range of extracellular matrix components (Inoue-Sakamoto et al.,

2018). It is necessary to determine if these molecules have the potential to inhibit PCR. In short,

this experiment involved utilizing three different NaPO4 buffer concentrations during a 500 µL

volume DNA extraction: 0%, 25%, and 50%. These percentages equate to 0 µL NaPO4/500 µL

sample, 125 µL NaPO4/375 µL sample, and 250 µL NaPO4/250 µL sample as the three volumes

added to a bead-beater tube, and each of these different assays are summarized as the numbers 1,

2, and 3, respectively, for simplicity. Following each M. aeruginosa pure culture DNA

extraction, the DNA was then diluted in TE buffer to obtain 1/10 (10-1), 1/100 (10-2), 1/1000 (10-

3), and 1/10,000 (10-4) dilutions from the original 100 extract. The results in Table 6 show all

three assays (1, 2, and 3) and Cts resulting from each dilution of that original DNA extract.

61

Table 6. DNA extraction buffer concentration effects on Ct. Each Sample Name starting with 1-, 2-, or 3- represents three different DNA extraction protocols, and Cts resulting from universal bacterial primers and probes according to each dilution. The sample used for DNA extraction was an M. aeruginosa pure culture (n=1).

Sample Name

Detector Name

Reporter Ct IPC Ct

NTC UnivP FAM Undetermined 26.8

NTC UnivP FAM Undetermined 26.7

1-100 UnivP FAM 36.6 26.5

1-100 UnivP FAM 37.5 26.3

1-10-1 UnivP FAM Undetermined 26.7








2-100 UnivP FAM 36.2 26.9

2-100 UnivP FAM 36.2 26.5

2-10-1 UnivP FAM 37.7 26.6

2-10-1 UnivP FAM 38 26.3







3-100 UnivP FAM 34.7 26.5

3-100 UnivP FAM 35.3 26.6

3-10-1 UnivP FAM 38.1 26.4

3-10-1 UnivP FAM 38.9 26.5







C diff-2 UnivP FAM 21.4 26.6

62

C diff-2 UnivP FAM 22.1 26.7

NAC IPC VIC N/A Undetermined

NAC IPC VIC N/A Undetermined

The results show that assay #3 (50% NaPO4/50% sample) has slightly lower Cts than assay #2

(25% NaPO4/75% sample). Using the Internal Positive Control protocol discussed in Section

2.5.1, no inhibition was observed. Table 6 shows the results of the IPC assay applied to the exact

same samples used in Table 5. The only sample that did not amplify with a Ct of 26 was the No

Amplification Control, as expected. These results suggest that the negative results in Table 5

were not the result of inhibition, but were below the limit of detection.

A second approach to evaluating inhibition caused by humic or fulvic acids in lake water was

performed using Synechococcus elongatus as a control. DNA was extracted from 250 µL of an S.

elongatus culture (+250 µL NaPO4 buffer), diluted 1/10, and labeled S-1. Additionally, 250 µL

of S. elongatus was combined with 250 µL of lake water, and in another tube, 250 µL of S.

elongatus was combined with 250 µL of molecular-grade water. DNA was extracted from both

of those tubes, and the DNA was diluted 1/10. A QPCR assay was performed using universal

cyanobacterial primers and probes, with the results shown in Table 7.

Table 7. Comparison of lake water + S. elongatus, molecular-grade water + S. elongatus, and S. elongatus pure culture dilutions, n=1 (LS-1 = Lake water + S. elongatus 10-1 dilution, MS-1 = Molecular-grade water + S. elongatus 10-1 dilution, S-1 = S. elongatus pure culture 10-1 dilution, Cdiff -3 = C. difficile 10-3 dilution, NTC = No Template Control).

Sample Name

Detector Name

Reporter Ct



LS-1 CYANO FAM 18.0

63

LS-1 CYANO FAM 18.0

MS-1 CYANO FAM 19.1

MS-1 CYANO FAM 19.0

S-1 CYANO FAM 20.4

S-1 CYANO FAM 20.3

Cdiff-3 CYANO FAM Undetermined


The culture with the lowest Ct was lake water + S. elongatus, possibly implying the presence of

additional cyanobacteria in the lake water.

3.2.3. Synechococcus elongatus-specific QPCR

This species is classified as picocyanobacteria, or small enough that it does not form visible algal

blooms (Callieri et al., 2012). Most water authorities are alerted to an algal bloom after visually

identifying it, so cyanobacteria that can grow in large numbers but evade visual detection, while

still producing toxins are a significant concern. An attempt was made to specifically detect S.

elongatus, but the Synechococcus-specific primers and probes did not produce a positive result

compared to universal primers and probes, which did produce a positive result (data not shown).

3.2.4. Lake Water Cyanobacteria Detection

Although lake water cultures amended with growth media produced visible cells, it was not

known if this growth was cyanobacteria, and might consist of eukaryotic algae and/or other

phyla of bacteria. The QPCR assay described in 3.2.2. also included lake water only, and

cyanobacteria were detected (Table 8).

64

Table 8. Cyanobacteria detected in Lake Mead water using universal cyanobacterial primers and probes, n=1 (L-1 = lake water DNA 10-1 dilution, Cdiff-3 = C. difficile 10-3 dilution, NTC = No Template Control, S-1 = S. elongatus 10-1 dilution).

Sample Name

Detector Name

Reporter Ct

L-1 CYANO FAM 38.0

L-1 CYANO FAM 37.2





S-1 CYANO FAM 20.4

S-1 CYANO FAM 20.3

3.2.5. Raphidiopsis raciborskii-specific QPCR

No BMAA-specific genes are currently known, but a QPCR assay was tested as a detection

method for Raphidiopsis raciborskii using primers and probes designed to detect

Cylindrospermopsis raciborskii. Table 9 shows that Raphidiopsis (RR) was successfully

amplified, and Microcystis (MA) was not.

Table 9. R. raciborskii primers and probes specificity, n=1 (NTC = No Template Control, RR-1 = R. raciborskii 10-1 dilution, MA-1 = M. aeruginosa 10-1 dilution, Cdiff-2 = C. difficile 10-2 dilution).

Sample Name

Detector Name

Reporter Ct

NTC cyrJ207 FAM Undetermined

NTC cyrJ207 FAM Undetermined

RR-1 cyrJ207 FAM 25.2

RR-1 cyrJ207 FAM 25.1

MA-1 cyrJ207 FAM Undetermined

MA-1 cyrJ207 FAM Undetermined

65

Cdiff-2 cyrJ207 FAM Undetermined

Cdiff-2 cyrJ207 FAM Undetermined

3.2.6. Microcystin-Production Genes

Three target genes were selected: mcyB, mcyD, and mcyE. Using DNA from non-toxin producing

M. aeruginosa from UTEX, and toxin-producing M. aeruginosa from the Bigelow National

Laboratory, it was possible to determine the specificity of each of the microcystin-production

genes by performing a QPCR assay with both of the DNA extracts. No amplification was

observed in QPCR assays that used mcyB-specific primers and probes. Tables 10 and 11 show

the use of primer/probe combinations for mcyD and mcyE specifically amplify the toxin-

producing Microcystis aeruginosa strain (MAT), and not the non-toxin producing Microcystis

aeruginosa strain (MA).

Table 10. mcyD primer/probe comparison of toxin-producing and non-toxin-producing M. aeruginosa, n=1 (MA-1 = M. aeruginosa 10-1 dilution, MAT-1 = M. aeruginosa toxin-producing 10-1 dilution, Cdiff-3 = C. difficile 10-3 dilution, NTC = No Template Control).

Sample Name

Detector Name

Reporter Ct

NTC mcyD FAM Undetermined

NTC mcyD FAM Undetermined

MA-1 mcyD FAM Undetermined

MA-1 mcyD FAM Undetermined

MAT-1 mcyD FAM 33.5

MAT-1 mcyD FAM 33.4

Cdiff-3 mcyD FAM Undetermined

66

Table 11. mcyE primer/probes comparison of toxin-producing and non-toxin-producing M. aeruginosa, n=1 (MA-1 = M. aeruginosa 10-1 dilution, MAT-1 = M. aeruginosa toxin-producing 10-1 dilution, Cdiff-3 = C. difficile 10-3 dilution, NTC = No Template Control).

Sample Name

Detector Name

Reporter Ct

NTC mcyE FAM Undetermined


MA-1 mcyE FAM Undetermined

MA-1 mcyE FAM Undetermined

MAT-1 mcyE FAM 36.8

MAT-1 mcyE FAM 38.8

Cdiff-3 mcyE FAM Undetermined

Cdiff-3 mcyE FAM Undetermined

3.2.7. mcyE QPCR Standard Curve

A sample was taken from a 200 mL BG-11 + 5 mL M. aeruginosa Kützing pure culture, and a

cell count was performed according to the protocol in Section 2.2.5. Particles between 2 and 5

µm were counted with a mean of 48,545 cells per 50 µL for three separate counts, at a dilution

factor of 20. The resulting mean cell count was 1.94E+07 cells/mL. The mean OD600 (n=5) of the

sample was 0.328. Serial dilutions of the sample were prepared and the OD600 was measured

(Table 12).

Table 12. Optical density measurements corresponding with cell counts.

OD600 Cell Count (cells/mL)

Cell Count (cells/µL)

0.328 1.94E+07 1.94E+04

0.033 1.94E+06 1.94E+03

0.005 1.94E+05 1.94E+02

67

Using a 3-point standard curve, the OD600 and cell count can be modeled using the following

equation in Figure 22.

Figure 22. Estimated 30-day growth curve relating cell count and OD600.

DNA extraction was performed on each of these samples, with 250 uL of sample used, resulting

in Table 13 showing the estimated number of cells present to provide DNA:

Table 13. Estimated number of cells present in each cell count culture DNA extraction.

Cell Count (cells/µL)

Volume (µL) # cells

19,400 250 4.85E+06

1,940 250 4.85E+05

194 250 4.85E+04

y = 6E+07x - 62532R² = 1

0.00E+00

5.00E+06

1.00E+07

1.50E+07

2.00E+07

2.50E+07

0 0.05 0.1 0.15 0.2 0.25 0.3 0.35

Ce

ll C

ou

nt

(ce

lls/m

L)

OD600

68

Following DNA extraction, and assuming one copy of the mcyE gene per cell, the following

number of mcyE genes is present in a 100 µL DNA extract, as shown in Table 14.

Table 14. Estimated mcyE/µL in the final DNA extract.

# cells Volume (µL) mcyE/µL

4.85E+06 100 4.85E4

4.85E+05 100 4.85E3

4.85E+04 100 4.85E2

A volume of 11 µL DNA extract is then added to a Mastermix volume of 44 µL, resulting in the

estimated number of mcyE genes per Mastermix (MM) tube, as shown in Table 15.

Table 15. Estimated cells per QPCR reaction well for the cell count culture.

Cells/µL Volume

(µL) mcyE/MM tube New mcyE/µL mcyE/well

4.85E+04 11 5.34E+05 9700 2.43E+05

4.85E+03 11 5.34E+04 970 2.43E+04

4.85E+02 11 5.34E+03 97 2.43E+03

For each of the cell count cultures, a Ct was obtained that corresponds to each sample, as shown in Table 16.

Table 16. Cts associated with cell count (CC) dilutions.

Sample Name

Detector Name Ct mcyE/well

CC 10^0 mcyE 26.4 2.43E+05

CC 10^0 mcyE 26.3 2.43E+05

69

CC 10^-1 mcyE 30.6 2.43E+04

CC 10^-1 mcyE 30.3 2.43E+04

CC 10^-2 mcyE 34.5 2.43E+03

CC 10^-2 mcyE 34.7 2.43E+03

Graphing Ct vs. Cell Count results in the following standard curve in Figure 23:

Figure 23. A standard curve relating # cells in a QPCR reaction well and resulting Ct.

3.3. Microcystin Production

Objective 3. Quantify toxin production from the surrogate system.

Toxin analysis was performed at the River Mountain Water Treatment Facility using LC-MS/MS

to analyze samples for the measurement of microcystin, anatoxin-a, cylindrospermopsin, and

y = -4.1x + 48.481R² = 1

15

20

25

30

35

40

0 1 2 3 4 5 6 7 8

Ct

log(X) Cell Count (# cells)

Standard Curve

Linear (Standard Curve)

70

nodularin toxins above a threshold of 0.5 µg/L. Among the dozens of known microcystin

congeners, the standards are available to measure the concentration of the following 12 specific

types: Microcystin-HilR, Microcystin-HtyR, Microcystin-LA, Microcystin-LF, Microcystin-LR,

Microcystin-LW, Microcystin-LY, Microcystin-RR, Microcystin-WR, Microcystin-YR,

Microcystin-dmLR, and Microcystin-dmRR. The most-studied congener is Microcystin-LR. The

first toxin-producing M. aeruginosa culture grown in the UNLV Emerging Diseases Laboratory

was sampled to evaluate toxin concentrations (Table 17):

Table 17. Toxin concentrations measured from M. aeruginosa pure culture used to inoculate other cultures.

Cyanotoxin Concentration

(µg/L)

Anatoxin-a <0.50

Cylindrospermopsin <0.50

Microcystin-HilR 5.1

Microcystin-HtyR <0.50

Microcystin-LA <0.50

Microcystin-LF 29

Microcystin-LR 190

Microcystin-LW 34

Microcystin-LY 14

Microcystin-RR <0.50

Microcystin-WR 1.7

Microcystin-YR <0.50

Microcystin-dmLR 31

Microcystin-dmRR <0.50

Nodularin <0.50

71

A volume of approximately 5 mL was extracted from this toxin-producing culture and inoculated

into a volume of approximately 200 mL BG-11 media. A sample was taken immediately from

this new culture and analyzed for toxins (Table 18).

Table 18. Comparison of estimated and actual toxin concentrations when inoculating a new culture.

Cyanotoxin Concentration (µg/L) Predicted Conc. Actual Conc.

Anatoxin-a <0.50 <0.50 <0.50

Cylindrospermopsin <0.50 <0.50 <0.50

Microcystin-HilR 5.1 <0.50 <0.50

Microcystin-HtyR <0.50 <0.50 <0.50

Microcystin-LA <0.50 <0.50 <0.50

Microcystin-LF 29 0.73 <0.50

Microcystin-LR 190 4.75 3.60

Microcystin-LW 34 0.85 0.52

Microcystin-LY 14 <0.50 <0.50

Microcystin-RR <0.50 <0.50 <0.50

Microcystin-WR 1.7 <0.50 <0.50

Microcystin-YR <0.50 <0.50 <0.50

Microcystin-dmLR 31 0.78 0.55

Microcystin-dmRR <0.50 <0.50 <0.50

Nodularin <0.50 <0.50 <0.50

The match between estimated and actual toxin concentrations suggests that there are no issues

with the toxin becoming lost during the LC-MS/MS process, and that the cells do not appear to

become stressed and release intracellular toxins during transport from UNLV to the River

Mountain Water Treatment facility.

72

Although toxin samples are expensive and time-consuming to process, toxin samples were taken

approximately once per week from this original Culture #1 culture, and the results of those toxin

samples are detailed in Table 19.

Table 19. 30-day toxin production, 200 mL BG-11 + 5 mL M. aeruginosa pure culture.

Cyanotoxin Day 0 (µg/L)

Day 5 Day 10 Day 16 Day 23 Day 30

Anatoxin-a <0.50 <0.50 <0.50 <0.50 <0.50 <0.50

Cylindrospermopsin <0.50 <0.50 <0.50 <0.50 <0.50 <0.50

Microcystin-HilR <0.50 <0.50 0.8 1.3 2.9 3.2

Microcystin-HtyR <0.50 <0.50 <0.50 <0.50 <0.50 <0.50

Microcystin-LA <0.50 <0.50 <0.50 <0.50 <0.50 <0.50

Microcystin-LF <0.50 1.50 7 12 16 18

Microcystin-LR 3.60 12.00 43.00 82.00 130.00 140.00

Microcystin-LW 0.52 1.90 14.00 24.00 26.00 22.00

Microcystin-LY <0.50 1.40 5 8 8 5

Microcystin-RR <0.50 <0.50 <0.50 <0.50 <0.50 <0.50

Microcystin-WR <0.50 <0.50 <0.50 <0.50 0.5 0.9

Microcystin-YR <0.50 <0.50 <0.50 <0.50 <0.50 <0.50

Microcystin-dmLR 0.55 1.10 3.90 10.00 38.00 50.00

Microcystin-dmRR <0.50 <0.50 <0.50 <0.50 <0.50 <0.50

Nodularin <0.50 <0.50 <0.50 <0.50 <0.50 <0.50

Microcystin-LR was produced in the greatest concentration over 30 days. Toxin samples were

taken from a culture consisting of 100 mL BG-11, 100 mL lake water, and 5 mL M. aeruginosa

inoculum. The total volume was the same as the previous culture (205 mL), but the available

nutrients were 50% compared to 200 mL BG-11. In addition, the lake water provides competitors

to M. aeruginosa for the available nutrients. Table 20 shows the results of these toxin

measurements.

73

Table 20. 60-day toxin concentrations from a 100 mL BG-11, 100 mL lake water, 5 mL M. aeruginosa culture.


Day 25 Day 60

Anatoxin-a <0.50 <0.50 <0.50


Microcystin-HilR <0.50 <0.50 <0.50



Microcystin-LF <0.50 2.20 2

Microcystin-LR 1.30 38.00 40.00

Microcystin-LW <0.50 3.20 3.40

Microcystin-LY <0.50 1.90 2


Microcystin-WR <0.50 <0.50 <0.50


Microcystin-dmLR <0.50 4.70 3.70


Nodularin <0.50 <0.50 <0.50

The 25-day Microcystin-LR concentration of 38 µg/L is approximately 30% of the 23-day

concentration of 130 µg/L in the original pure culture (Table 19), so the toxin production in the

BG-11/lake water culture is significantly less than 50%. Additionally, when the experiment was

carried out to 60 days, the toxin concentrations were basically unchanged given an extra month

of growth.

An experiment was performed to provide even less growth media availability with a culture

consisting of 200 mL lake water, 40 mL BG-11, and 5 mL M. aeruginosa inoculum (Table 21).

74

Table 21. Toxin production in 200 mL lake water + 40 mL BG-11 + 5 mL M. aeruginosa.


Day 10

Day 15

Day 22

Day 29

Day 36

Day 42

Day 50

Day 57

Anatoxin-a <0.50 <0.50 <0.50 <0.50 <0.50 <0.50 <0.50 <0.50 <0.50 Cylindrospermopsin <0.50 <0.50 <0.50 <0.50 <0.50 <0.50 <0.50 <0.50 <0.50

Microcystin-HilR <0.50 <0.50 <0.50 <0.50 <0.50 <0.50 <0.50 <0.50 <0.50 Microcystin-HtyR <0.50 <0.50 <0.50 <0.50 <0.50 <0.50 <0.50 <0.50 <0.50 Microcystin-LA <0.50 <0.50 <0.50 <0.50 <0.50 <0.50 <0.50 <0.50 <0.50 Microcystin-LF <0.50 <0.50 <0.50 <0.50 <0.50 <0.50 <0.50 <0.50 <0.50 Microcystin-LR 2.50 <0.50 <0.50 <0.50 <0.50 <0.50 <0.50 <0.50 <0.50 Microcystin-LW <0.50 <0.50 <0.50 <0.50 <0.50 <0.50 <0.50 <0.50 <0.50 Microcystin-LY <0.50 <0.50 <0.50 <0.50 <0.50 <0.50 <0.50 <0.50 <0.50 Microcystin-RR <0.50 <0.50 <0.50 <0.50 <0.50 <0.50 <0.50 <0.50 <0.50 Microcystin-WR <0.50 <0.50 <0.50 <0.50 <0.50 <0.50 <0.50 <0.50 <0.50 Microcystin-YR <0.50 <0.50 <0.50 <0.50 <0.50 <0.50 <0.50 <0.50 <0.50

Microcystin-dmLR <0.50 <0.50 <0.50 <0.50 <0.50 <0.50 <0.50 <0.50 <0.50 Microcystin-dmRR <0.50 <0.50 <0.50 <0.50 <0.50 <0.50 <0.50 <0.50 <0.50

Nodularin <0.50 <0.50 <0.50 <0.50 <0.50 <0.50 <0.50 <0.50 <0.50

The original concentration of 2.5 µg/L Microcystin-LR was detected in the Day 0 sample.

However, the disappearance of that measurable amount of Microcystin-LR, and with no toxins

detected during weekly sampling over 60 days was unexpected.

The 200 mL lake water + 40 mL BG-11 + 5 mL M. aeruginosa culture was repeated, with the

toxin results shown in Table 22.

75

Table 22. Toxin production in 200 mL lake water + 40 mL BG-11 + 5 mL M. aeruginosa repeated experiment.


Day 21 Day 58

Anatoxin-a <0.50 <0.50 <0.50


Microcystin-HilR <0.50 <0.50 <0.50



Microcystin-LF <0.50 <0.50 <0.50

Microcystin-LR <0.50 0.77 0.70

Microcystin-LW <0.50 <0.50 <0.50

Microcystin-LY <0.50 <0.50 <0.50


Microcystin-WR <0.50 <0.50 <0.50


Microcystin-dmLR <0.50 <0.50 <0.50


Nodularin <0.50 <0.50 <0.50

Compared with previous Day 0 samples from other cultures, there were no toxins initially

detected from the M. aeruginosa that was inoculated into the 200 mL lake water + 40 mL BG-11

media. However, a low concentration of Microcystin-LR was detected in samples taken at Day

21 and Day 58.

When mcyE Ct was compared between an M. aeruginosa pure culture grown in 200mL BG-11

and a culture with reduced media (40mL BG-11), the Ct associated with little to no microcystin

is still Ct 33, suggesting that Ct and toxin concentration are not directly correlated. Table 23

summarizes this comparison.

76

Table 23. Comparison of pure culture (200mL BG-11) and reduced media (40mL BG-11) Ct values at approximately 60 days, assuming one copy of mcyE per cell.

Time (days) Pure Culture

Microcystin LR (ug/L)

mcyE CT mcyE/well Cells/mL

0 3.6 35.6 1.39E+03 5.56E+04

58 160 27.7 1.17E+05 4.68E+06

Time (days) Reduced Media Microcystin LR

(ug/L) mcyE CT mcyE/well Cells/mL

0 0 Undetermined < 1E+02 < 5E+03

58 0.7 33.8 3.81E+03 1.52E+05

3.4. BMAA Production by Raphidiopsis raciborskii

Objective 4: Study the production of other cyanotoxins

3.4.1. Anatoxin-a

Cultures were typically maintained for much longer than 30-60 days in order to assess the

maximum amount of toxins a culture can produce given finite nutrient availability. A culture

consisting of 200 mL BG-11, 200 mL autoclaved lake water, and 5 mL M. aeruginosa gave a

positive result for the detection of anatoxin-a on day 104 as shown in Table 24.

Table 24. Detection of Anatoxin-a in 200 mL BG-11 + 200 mL autoclaved lake water + 5 mL M. aeruginosa inoculum.


Anatoxin-a 3.10

Cylindrospermopsin <0.50

Microcystin-HilR 16

Microcystin-HtyR <0.50

77

Microcystin-LA <0.50

Microcystin-LF 21

Microcystin-LR 380

Microcystin-LW 17

Microcystin-LY 3

Microcystin-RR <0.50

Microcystin-WR 9.4

Microcystin-YR <0.50

Microcystin-dmLR 190

Microcystin-dmRR <0.50

Nodularin <0.50

3.4.2. Cylindrospermopsin

Although the toxin of interest with regards to Raphidiopsis raciborskii was BMAA, it was

observed that this culture produces cylindrospermopsin. At day 14, 170 µg/L of

cylindrospermopsin was measured (data not shown).

3.4.3. BMAA

3.4.3.1. BMAA produced by R. raciborskii

Cultures were inoculated in order to assess BMAA production by R. raciborskii. The culture

used as inoculum for each of the cultures originated from the 14-day culture in Section 3.4.2 that

was found to produce cylindrospermopsin. After attempting to grow R. raciborskii in four

different types of media (Modified Combo, BG-11, Jaworski’s, and DYV), the culture growing

in Modified Combo media had excellent growth that was confirmed microscopically by the

presence of filamentous cells. Similar to the concept of adding 5 mL M. aeruginosa to 200 mL of

growth media to establish a fresh culture, 1.25 mL of R. raciborskii was inoculated into 50 mL of

Modified Combo media. A volume of 1.25 mL of R. raciborskii was also inoculated into 50 mL

of lake water without growth media. To determine how volume affects growth and toxin

78

production, a culture consisting of 100 mL Modified Combo media and 2.5 mL R. raciborskii

inoculum was prepared, doubling the 50 mL Combo + 1.25 mL R. raciborskii culture. A culture

with 50 mL Modified Combo media, 50 mL lake water, and 2.5 mL R. raciborskii inoculum was

also prepared. The 30-day growth curves associated with each of these cultures are shown in Fig

23.

Figure 23. Comparison of pure culture and lake water-amended R. raciborskii growth curves.

0.001

0.01

0.1

1

0 5 10 15 20 25 30

OD

60

0

Time (days)

R. raciborskii Growth Curves

50mL Combo + 1.25mL R.r.

50mL Combo + 50mL lake H2O +2.5mL R.r.

50mL lake H2O + 1.25mL R.r.

100mL Combo + 2.5mL R.r.

79

For each of these OD600 measurements, samples were analyzed for BMAA production. Although

the literature suggests that R. raciborskii produces large quantities of free BMAA, this was not

observed, as shown in the following summary of results:

50 mL Modified Combo + 1.25 mL R. raciborskii

1/7/20 – BMAA = < 5.0 ug/L

1/21/20 – BMAA = < 5.0 ug/L

3/4/20 – BMAA = 5 ug/L

50 mL Modified Combo + 50 mL Lake Water + 2.5 mL R. raciborskii

12/31/19 – BMAA = < 5.0 ug/L

1/21/20 – BMAA = 6.1 ug/L

3/4/20 – BMAA = < 5.0 ug/L

50 mL Lake Water + 1.25 mL R. raciborskii

1/7/20 – BMAA = < 5.0 ug/L

1/21/20 – BMAA = < 5.0 ug/L

3/4/20 – BMAA = < 5.0 ug/L

100 mL Modified Combo + 2.5 mL R. raciborskii

1/7/20 – BMAA = < 5.0 ug/L

1/21/20 – BMAA = 6.1 ug/L

3/4/20 – BMAA = < 5.0 ug/L

3.4.3.2. BMAA in Drinking Water

Due to the fact that BMAA in drinking water is not federally regulated, the unique access to

BMAA analysis at the River Mountain Water Treatment facility offered a unique chance to

address the following question:

- Is BMAA present in Las Vegas drinking water?

80

A Las Vegas tap water sample was analyzed for BMAA, resulting in undetectable levels (< 5.0

µg/L). Additionally, a sampling vial was filled directly from the tap at a sink in a UNLV

bathroom. Again, the results of this test were undetectable levels of BMAA (< 5.0 µg/L, n=2).

Water is commonly consumed in Las Vegas from water bottles purchased at the store, so random

water bottles were also tested for the presence of BMAA. First, a bottle of water purchased from

a Las Vegas gas station was sampled, and BMAA was undetectable (< 5.0 µg/L). A bottle of

water from UNLV was tested, and BMAA was again undetectable in that sample (< 5.0 µg/L,

n=2). The Lake Mead water provided by the Southern Nevada Water Authority was also

sampled, resulting in undetectable BMAA (< 5.0 µg/L, n=2).

3.4.3.3. BMAA in Seafood

The presence of BMAA in seafood has the potential to be an emerging threat to global public

health. For this reason, seafood was tested in this study as a presence/absence control in

comparison to the R. raciborskii samples. The following BMAA measurements were reported

from seafood samples:

- 1/21/20 Wild caught cod, China, BMAA = Present

- 3/4/20 – Wild caught cod, USA, BMAA = Present

- 3/4/20 – Salmon (farm-raised), BMAA = Present

- 3/4/20 – Shrimp (farm-raised) BMAA = Present

- 3/4/20 – Tuna (product of Thailand) BMAA = Present

- 3/4/20 – RO water control, BMAA = Absent

81

3.4.3.4. BMAA in Diverse Cyanobacterial Species

The literature suggests that BMAA is produced by most cyanobacterial species. In order to test

this assertion, the wide variety of cyanobacterial species growing in the laboratory at UNLV

were evaluated to determine if BMAA production is common. Similar to the BMAA detected in

seafood, these values are not quantitative. As described in Chapter 2, some cyanobacteria clump

together and are very difficult to manually separate. In these situations, some media that had

been in contact with clumps of cells was used to sample for BMAA production. The following

results were obtained:

1/21/20 Microcystis aeruginosa BMAA = Present

3/4/20 Microcystis aeruginosa BMAA = Present

1/21/20 Synechococcus elongatus BMAA = Absent

3/4/20 Synechococcus elongatus BMAA = Absent

1/21/20 Nodularia spumigena BMAA = Present

3/4/20 Nodularia spumigena BMAA = Absent

1/21/20 Anabaena flos-aquae BMAA = Present

3/4/20 Anabaena flos-aquae BMAA = Absent

1/21/20 Oscillatoria lutea BMAA = Present

3/4/20 Oscillatoria lutea BMAA = Absent

1/21/20 Lyngbya aestuarii BMAA = Present

3/4/20 Lyngbya aestuarii BMAA = Present

82

1/21/20 Nostoc commune BMAA = Present

3/4/20 Nostoc commune BMAA = Present

1/21/20 Aphanizomenon BMAA = Absent

3/4/20 Aphanizomenon BMAA = Present

3.6. Growth Potential from Soil Fertilizer

Stormwater is a major source of nutrients in Lake Mead, as untreated stormwater is discharged

directly to the lake through the Las Vegas Wash. In general, Las Vegas weather can be described

by periods of little to no rain, which allows for oil, grease, herbicides, pesticides, and fertilizer

nutrients to accumulate in the soil, followed by a period of intense rain, and subsequent polluted

urban runoff. In order to simulate a stormwater event, fertilizer purchased at Walmart was

introduced to samples of Lake Mead water. As an initial experiment, 1 g of time-release fertilizer

pellets were added to 75 mL of lake water and incubated under 12 hr light/dark cycles. The flask

developed thick green growth. A second culture was prepared with 1 g of autoclaved fertilizer

pellets, but this flask did not produce any growth visible to the naked eye. These results suggest

that microbes present on the fertilizer pellets were responsible for the growth observed in the

flask with non-autoclaved fertilizer. Samples were taken for toxin analysis from the flask with

visible growth, and although no microcystin, anatoxin-a, cylindrospermopsin, or nodularin were

detected, a BMAA concentration of 25 µg/L was detected. This is in contrast to lake water

samples that do not have detectable BMAA.

3.7. Comparison of DNA Extraction Methods

The Amicon Ultra DNA purification and concentration method was used in this study. With a

focus on producing a rapid QPCR protocol, a freeze-thaw protocol was tested as discussed in

83

Section 2.4.2. A pure culture of M. aeruginosa was used for both standard DNA extraction and

freeze-thaw DNA extraction, with the results shown in Table 25.

Table 25. Difference in Ct between standard DNA extraction (EXTR) and freeze-thaw DNA extraction (HS). MAT10X is a 10X concentrated M. aeruginosa positive control, NTC is a No Template Control, n=1.

Sample Name

Detector Name Reporter Ct

EXTR mcyE FAM 27.8

EXTR mcyE FAM 27.9

HS mcyE FAM 22.2

HS mcyE FAM 22.6

MAT10X mcyE FAM 25.1




This approximately 5 Ct difference between extracts from the same sample was not expected, so

the experiment was repeated using cells that had been 10x concentrated by centrifugation, and

also using a different sample taken from a 100 mL lake water + 100 mL BG-11 + 5 mL M.

aeruginosa culture, as shown in Table 26.

84

Table 26. Difference in Ct between standard DNA extraction (10XExtr) and freeze-thaw DNA extraction (10XHS), n=1.

Sample Name Detector Reporter Ct

10XExtr mcyE FAM 37.92

10XExtr mcyE FAM 38.04

10XHS mcyE FAM 28.48

10XHS mcyE FAM 33.05

Overall, it is observed from Table 26 that freeze-thaw DNA extraction resulted in lower Ct

values than standard DNA extraction.

3.8. Blinded Experiment

A blinded experiment was conducted to assess the ability to differentiate between toxin-

producing and non-toxin-producing samples. In short, a laboratory member was given a culture

consisting of visible cell growth from 100 mL lake water + 100 mL BG-11 + 5 mL M.

aeruginosa, which had been previously shown to produce Microcystin-LR of 40 µg/L. The

laboratory member was also given a flask containing visible cell growth from 50 mL lake water

+ 50 mL BG-11, which had been previously shown to have undetectable Microcystin-LR

production (<0.5 µg/L). In isolation, the laboratory member aliquoted the toxin-producing

culture into a 15 mL Falcon tube, and two aliquots from the non-toxin-producing culture into two

separate 15 mL Falcon tubes, for a total of three samples, labeled A, B, and C. Although the

visible growth in the toxin-producing bottle was a somewhat similar green color to the growth in

the non-toxin-producing bottle, each aliquot was wrapped in aluminum foil to make the

experiment truly blinded. The laboratory member was the only person who knew which tube, A,

B, or C, contained the toxin-producing culture. The freeze-thaw protocol was then applied to the

85

samples as described in Section 2.4.2, and TaqMan QPCR was performed targeting the mcyE

gene. Negative PCR results were obtained for all three cultures.

Using a different approach, a volume of 2.5 mL was concentrated 10-fold by centrifugation and

re-suspended in 250 µL molecular-grade water. The freeze-thaw DNA extraction protocol was

then applied to this 10X concentrated sample and PCR was conducted (Table 27).

Table 27. Blinded PCR results using 10X concentrated cells, compared to a 10X positive control (MAT10X = M. aeruginosa toxin-producing culture 10X concentrated positive control, NTC = No Template Control).

Sample Name

Detector Name Reporter Ct

A blind mcyE FAM Undetermined

A blind mcyE FAM Undetermined

B blind mcyE FAM Undetermined

B blind mcyE FAM Undetermined

C blind mcyE FAM 33.2

C blind mcyE FAM 33.0





The laboratory member confirmed that indeed, the toxin-producing sample was Tube C. This

protocol, with the addition of a 10X concentration step, was able to identify the toxin-producing

sample in a completely blinded experiment.

3.9. Microcystin-Detection Protocol Results

Objective 5. Develop a protocol for water quality control/management.

86

The protocol described in Section 2.7 separates the detection of mcyE genes into two different

categories:

1. Samples taken from visible surface cell accumulation

2. Samples taken with no visible surface cell accumulation

The difference between these two sample types is that Sample Type 1 does not require a 10X

concentration, but Sample Type 2 does require this extra step.

The results of the sample taken from a surface cell accumulation in the laboratory are given in

Table 28.

Table 28. PCR results from a surface cell accumulation (Sample) compared with a M. aeruginosa toxin-producing pure culture (Positive Control).

Well # Sample Name Detector Name Ct Cells/mL

1 NTC mcyE Undetermined < 5E+03


25 Sample mcyE 23.6 4.68E+07

26 Sample mcyE 23.9 3.96E+07

59 Positive Control mcyE 25.1


In the second scenario, a culture lacking surface cell accumulation, which had M. aeruginosa

inoculated into the culture, but produces undetectable microcystin concentrations (MC+), was

compared to a similar culture that did not have M. aeruginosa inoculated into the culture (MC-).

Without the 10X concentration step, the sample would not produce a Ct, giving a false negative

result. The Ct results are shown in Table 29.

87

Table 29. 10X concentrated results showing detection of a culture containing M. aeruginosa (MC+), compared to a culture lacking M. aeruginosa.

Well Sample Name Detector Name Ct Cells/mL



25 MC+ mcyE 39.8 5.24E+03

26 MC+ mcyE 39.2 7.34E+03

27 MC- mcyE Undetermined < 5E+03

28 MC- mcyE Undetermined < 5E+03


60 Positive Control mcyE 25

88

Chapter 4. Discussion and Conclusions

4.1. Objective 1. Establish a surrogate (simulation) system to grow M. aeruginosa in Lake Mead conditions.

4.1.1. Full-spectrum lighting and variable temperatures cultivate diverse cyanobacteria

The use of light emitting diodes (LEDs) in the cultivation of algae is not well represented in the

literature, as the traditional method has been the use of cold white fluorescent light (Duarte and

Costa, 2018; Sirisuk et al., 2018). The LED light used in this study was a full-spectrum blue-to-

red light, and these additional wavelengths have demonstrated improved pigment energy

utilization in algae compared to fluorescent lights (Schulze et al., 2014). The decision to use an

LED light as opposed to a traditional light source was a new approach with this project, and the

growth curve of M. aeruginosa closely matches other reported growth curves (Bortoli et al.,

2014). Future work that grows cultures in the same type of environmental chamber but with

different light sources is recommended to clearly identify if LED is superior, equivalent, or less

effective than the use of white fluorescent light when growing cyanobacteria in the laboratory.

4.1.2. BG-11 media cultivates toxin-producing M. aeruginosa

Another change in culture conditions in this study was the use of BG-11 media instead of other

commonly used media such as Bold 3N. As shown in Appendix V, the media recipes differ

significantly in trace metals used, citrate, sulfate, Vitamin B12, etc. Both can be used to grow

Microcystis aeruginosa, but this study focused only on growth using BG-11. Future work that

utilizes the same LED light in this study, but studies the differences between growth curves and

toxin production with these different media types is suggested. The low number of replicates

(n=1 to n=4) for each of the cultures is indicative of the space limitation within the

89

environmental chamber. It was not possible to perform this experiment with n=10 or more given

space limitations, and also the limitation of each assay lasting one month.

4.2. Objective 2. Use QPCR to quantify the density of M. aeruginosa from the surrogate system.

4.2.1. Microcystin-production gene primers and probes suggest specificity

One advantage of this study was the acquisition of both a toxin-producing M. aeruginosa culture

(Bigelow National Laboratory) and a non-toxin-producing M. aeruginosa culture (UTEX) for the

assessment of QPCR specificity. Researchers have been seeking to clarify this dilemma for quite

some time with regards to whether the difference between toxic and non-toxic strains is due to

gene content or down-regulation of toxin production in cells with the same gene content

(Meissner et al., 1996). The mcyE gene was selected for two reasons:

1. The literature suggests that Adda and D-Glu, which are synthesized by mcyE, are most

common components in 279 known microcystin structures compared to all other amino

acids (Bouaicha et al., 2019).

2. The length of the PCR amplicon is >100bp, compared to mcyD amplicons are <100bp,

and the protocol for cleaning PCR products for use in other experiments requires

amplicons >100bp.

For the purposes of this study, it was shown that mcyE was amplified in the toxin-producing

strain, and no amplification was observed in the non-toxin-producing strain.

4.2.2. M. aeruginosa limit of detection is approximately 5000 cells/mL

An important metric when designing a protocol for detecting an organism is to determine the

limits of detection. Given a sample (100), when diluted to 10-1, 10-2, and 10-3, the 10-3 sample has

90

been problematic with regards to obtaining a Ct value, typically resulting in Undetermined. The

lower limit of detection is estimated to be ~5000 cells/mL (4.68E+03 cells/mL) based on the

standard curve equation of the number of mcyE genes per well in Figure 22, assuming one copy

of the gene per cell to determine cells/mL. The assumption of one mcyE gene per cell has been

used before, although some studies have shown that this can provide incorrect cell abundance

estimates (Lee et al., 2015; Crosby and Criddle, 2003; Rinta-Kanto et al., 2009). Future work

should be performed on this culture to sequence the genome and determine the accurate mcyE

copy number per cell for this M. aeruginosa strain, and for any M. aeruginosa strain later

isolated from Lake Mead.

4.3. Objective 3. Quantify toxin production from the surrogate system.

4.3.1. Toxin concentration cannot be related to cell concentration.

The literature suggests that toxin gene concentration and microcystin production are not directly

correlated (Guedes et al., 2014; Beversdorf et al., 2015). This study supports those results, but

the results presented in this study are the first to identify an exact media concentration that still

allows for cell growth, but not for microcystin production. This provides insight into future work

to address water quality characteristics that promote or limit microcystin production.

4.3.2. A single species of M. aeruginosa produces multiple microcystin congeners

The LC-MS/MS assay is limited to the number of microcystin congeners available as positive

controls. Of the 12 congeners measured in this study, it is common for M. aeruginosa to produce

seven different microcystin toxins, with Microcystin-LR consistently measured as the most

abundant form. The literature suggests that microcystin composition and concentration is

influenced by nitrogen, which would explain why consistent use of the same growth media

91

results in the same profile of microcystin types produced (Monchamp et al., 2014). Future work

to adjust nitrogen concentrations in media would be an interesting approach to determining if

Microcystin-LR is always the most abundant form of microcystin, or if this is due to the nitrogen

content of BG-11 media.

4.3.3. Microcystin is easily transferred and does not bind with surfaces

When a sample was taken from an M. aeruginosa culture and evaluated for Microcystin-LR

concentrations, the measurement in µg/L allowed for the calculation of the µg of toxin present in

a well-mixed 200 mL culture. When 5 mL of that same culture was inoculated into a new flask, a

well-mixed sample was taken immediately to determine the initial toxin concentration.

Interestingly, the toxin concentrations measured from the new flask matched with the calculated

concentrations, which demonstrates that there are no issues with microcystin binding to cells, to

tubes, to pipettes, or lost during sample processing in LC-MS/MS.

4.3.4. Microcystin production is nutrient-dependent

In this study, microcystin production was shown to vary according to nutrient availability.

Compared to a pure culture standard in 200 mL of media, the amount of toxins produced when

100 mL of media was mixed with 100 mL of lake water was less than half of the toxin

production of the pure culture. It is not known if this reduction is due to resource competition

with organisms naturally present in the lake water, due to the reduction of available nutrients in

the media due to dilution, or a combination of both. A third experiment showed that cultures

containing 200 mL of water and 40 mL of media result in undetectable or nearly-undetectable

toxin concentrations. Future work will be necessary to determine exactly which nutrient

92

concentration eliminates toxin production, and that can become a baseline for measuring nutrient

concentrations in Lake Mead to supplement water quality monitoring approaches.

4.4. Objective 4. Study the production of other cyanotoxins.

4.4.1. R. raciborskii BMAA production is dependent on growth conditions

This culture, formerly referred to as Cylindrospermopsis raciborskii CR3, was specifically

obtained from Australia due to the literature showing that it is the most prolific producer of free

BMAA of all freshwater cyanobacteria (Cox et al., 2005). After attempting to grow this culture

with four different types of media, there was tremendous growth using Modified Combo media

(UTEX). There was a small amount of growth observed in BG-11 media, and no growth was

visible in DYV and Jaworski’s media. As demonstrated in Table 3, CuSO4 was the only

component of the media that matched the growth/no-growth results in this study. Additionally,

Modified COMBO media had more components than BG-11 media, which explains the

differences between those media types. However, the culture conditions and growth media were

never able to replicate the BMAA production observed in natural samples. This study did not

perform a dry weight (µg/g) evaluation of BMAA, but M. aeruginosa was found to produce 15

µg/L after 30+ days, compared to an average of 5 µg/g free BMAA in the Cox et al., 2005 study,

which gives a baseline of comparison with a maximum of 12 µg/L BMAA produced by the R.

raciborskii culture in the laboratory, compared to 6,478 µg/g detected in Australian freshwater.

Future work is necessary to try other media recipes and growth temperatures to assess if BMAA

production can be increased in the laboratory by changing growth conditions.

93

4.4.2. BMAA is not detected in Las Vegas drinking water

Water authorities are not required by federal law to test drinking water for BMAA. As a simple

presence/absence experiment, tap water samples were collected from a home in Las Vegas, and

from a bathroom sink at UNLV. An n=2 sample size is not satisfactory to make any significant

decisions about BMAA in drinking water in Las Vegas, but this analysis has not been previously

observed in the literature. Additionally, BMAA was not detected in two Lake Mead water

samples and two bottled water samples. However, the potential for BMAA production exists

because the sample that produced growth from a mixture of Lake Mead water and 1 g non-

autoclaved fertilizer did result in a significant amount of BMAA detected (25 µg/L). The source

of the organism responsible for producing the BMAA likely originated from the fertilizer pellets,

due to the lack of growth produced using autoclaved fertilizer pellets. Future work should be

performed to obtain a 16S rRNA gene survey of the fertilizer-induced growth to determine which

organisms are present, compared to a 16S rRNA gene survey of the lake water growth. This

would give insight into the microbe that is associated with fertilizer that produces BMAA using

only the nutrients available in fertilizer, which the lake water has not shown the ability to do.

4.4.3. BMAA in seafood is a potential public health concern

This portion of the project was also meant as a presence/absence survey of BMAA in seafood.

The results also supported the ability to assess abundance based on wild-caught vs. farm-raised

seafood products. BMAA was present in all seafood samples, and was also observed to be

abundant in wild-caught samples. The lack of quantitative association with these samples due to

not having a measurement of the mass of sample used, or volume of water introduced to the tube,

limits the conclusions that can be drawn from these data, but it is clear that this is a topic that

should be the subject of continuing investigation. Overall, the conclusion can be made that the

94

public consumes seafood, that BMAA is present and sometimes abundant in seafood, and

therefore toxins in seafood have the potential to be a public health concern.

4.5. Objective 5. Develop a protocol for water quality control/management.

4.5.1. Toxin-producing M. aeruginosa can be detected in a 75 minute assay

The presence of toxin-producing M. aeruginosa in Lake Mead, as confirmed by LC-MS/MS, is a

problem that water authorities encountered in 2015. Surface accumulations were visible in some

coves and marinas, and under the microscope the cells had the characteristic shape of M.

aeruginosa. Given these same conditions, with the 8-step PCR protocol described in Chapters 2

and 3, microcystin-production genes can be detected in 75 minutes or less, providing water

managers with information they can use to make decisions regarding public health. This is a

simple and rapid tool that can be performed by a single person, and significantly advances the

ability to detect microcystin-producing cyanobacteria. Given an active visual accumulation of

surface growth in Lake Mead, based on the results of simulated blooms in this study, samples

with an “Undetermined” mcyE Ct indicate that the bloom does not contain detectable

microcystin-producing cyanobacteria. Any measurable Ct value indicates that microcystin

monitoring should be performed. Additional work is required to further validate this protocol,

but this study has clearly outlined a format that has demonstrated effectiveness at detecting

toxin-producing Microcystis aeruginosa in simulated Lake Mead algal blooms, as summarized in

Figure 24.

95

Figure 24. Summary of a comprehensive plan for routine monitoring, sampling of visible surface accumulated growth, and lab monitoring.

4.6 Limitations of the Study

The limitations of this study are centered on three key constraints: the limited space available

under the LED light, the limited number of samples that could be tested for toxin concentrations,

and a low number of repeated PCR assays. These key parameters were significantly restricted,

resulting in low n values across all experiments, from low numbers of cultures to toxin samples

96

that were only relevant to presence/absence in a sample. These limitations must be resolved

through validation in future work. For example, the results of the significant differences between

DNA extraction protocols was unexpected, but has a major impact on the proposed rapid

detection protocol. Before moving forward with adopting either of the DNA extraction methods

presented in this study, it will be necessary to determine if the results showing freeze-thaw to be

a superior technique are valid. Prior to starting the study, it was hypothesized that cell

concentrations could be used to predict toxin concentrations as part of a rapid assay. This study

was unable to correlate these concentrations directly. Although the use of direct cell counting

provided an excellent method of establishing a standard curve in this study, it was only

performed once, and future work should be focused on repeating the same protocols detailed in

this study to validate the assay.

97

Appendix I – Growth Curves

Table 30. M. aeruginosa pure culture.

Table 31. M. aeruginosa 2nd pure culture.

Sample name Date Time Sample delta t (hrs) Time (days) OD600

200mL BG-11 + 5mL M.a. 8/18/2019 1605 0 0 0.013

200mL BG-11 + 5mL M.a. 8/23/2019 1125 115 4.8 0.028

200mL BG-11 + 5mL M.a. 8/28/2019 1810 127 10.1 0.107

200mL BG-11 + 5mL M.a. 9/3/2019 0940 136 15.7 0.171

200mL BG-11 + 5mL M.a. 9/10/2019 0940 168 22.7 0.212

200mL BG-11 + 5mL M.a. 9/17/2019 1055 169 29.8 0.287

200mL BG-11 + 5mL M.a. 9/24/2019 0845 166 36.7 0.305

200mL BG-11 + 5mL M.a. 10/1/2019 0735 167 43.6 0.358

200mL BG-11 + 5mL M.a. 10/8/2019 0755 168 50.7 0.423

200mL BG-11 + 5mL M.a. 10/15/2019 0925 170 57.7 0.427

200mL BG-11 + 5mL M.a. 10/21/2019 0940 144 63.7 0.443

200mL BG-11 + 5mL M.a. 10/29/2019 0615 189 71.6 0.575

200mL BG-11 + 5mL M.a. 11/5/2019 1055 173 78.8 0.704

200mL BG-11 + 5mL M.a. 11/12/2019 0855 166 85.7 0.817

200mL BG-11 + 5mL M.a. 11/19/2019 0935 169 92.7 0.714

200mL BG-11 + 5mL M.a. 11/26/2019 1005 169 99.8 0.873

200mL BG-11 + 5mL M.a. 12/3/2019 0835 167 106.7 0.839

200mL BG-11 + 5mL M.a. 12/11/2019 1230 196 114.9 0.974

200mL BG-11 + 5mL M.a. 12/17/2019 0855 140 120.7 1.059

200mL BG-11 + 5mL M.a. 12/31/2019 1825 346 135.1 1.277

200mL BG-11 + 5mL M.a. 1/7/2020 1050 160 141.8 1.461

200mL BG-11 + 5mL M.a. 1/23/2020 1320 387 157.9 1.844

200mL BG-11 + 5mL M.a. 2/10/2020 1110 430 175.8 1.916


200mL BG-11 + 5mL M.a. 10/22/2019 0945 0 0 0.022

200mL BG-11 + 5mL M.a. 10/29/2019 1005 168 7 0.063

200mL BG-11 + 5mL M.a. 11/5/2019 1110 169 14 0.097

200mL BG-11 + 5mL M.a. 11/12/2019 0910 166 21 0.145

200mL BG-11 + 5mL M.a. 11/19/2019 0945 169 28 0.132

200mL BG-11 + 5mL M.a. 11/26/2019 1015 169 35 0.211

200mL BG-11 + 5mL M.a. 12/3/2019 0845 167 42 0.218

200mL BG-11 + 5mL M.a. 12/11/2019 1240 196 50 0.371

200mL BG-11 + 5mL M.a. 12/17/2019 0905 140 56 0.270

200mL BG-11 + 5mL M.a. 12/31/2019 1835 346 70 0.241

200mL BG-11 + 5mL M.a. 1/7/2020 1100 160 77 0.263

200mL BG-11 + 5mL M.a. (+BG) 1/23/2020 1325 386 93 0.200

98

Table 32. M. aeruginosa 3rd pure culture.

Table 33. M. aeruginosa 4th pure culture.

Table 34. M. aeruginosa grown in lake water.

Table 35. M. aeruginosa 2nd culture grown in lake water.


200mL BG-11 + 5mL M.a. 11/8/2019 1840 0 0 0.005

200mL BG-11 + 5mL M.a. 11/12/2019 0925 87 4 0.012

200mL BG-11 + 5mL M.a. 11/15/2019 1550 78 7 0.065

200mL BG-11 + 5mL M.a. 11/19/2019 0955 90 11 0.145

200mL BG-11 + 5mL M.a. 11/26/2019 1025 169 18 0.129

200mL BG-11 + 5mL M.a. 12/3/2019 0855 167 25 0.160

200mL BG-11 + 5mL M.a. 12/11/2019 1300 196 33 0.176

200mL BG-11 + 5mL M.a. 12/17/2019 0915 140 39 0.178

200mL BG-11 + 5mL M.a. 12/31/2019 1845 346 53 0.199

200mL BG-11 + 5mL M.a. 1/7/2020 1115 160 60 0.354

200mL BG-11 + 5mL M.a. 1/23/2020 1335 386 76 0.352

200mL BG-11 + 5mL M.a. 2/10/2020 1130 430 94 0.367


200mL BG-11 + 5mL M.a. 1/9/2020 1200 0 0 0.005

200mL BG-11 + 5mL M.a. 1/23/2020 1345 338 14 0.180

200mL BG-11 + 5mL M.a. 2/10/2020 1140 430 32 0.224


200mL BG-11 + 200mL lake H2O + 5mL M.a. 8/18/2019 1635 0 0 0.005

200mL BG-11 + 200mL lake H2O + 5mL M.a. 8/23/2019 1120 115 4.8 0.013



















99

Table 36. M. aeruginosa grown in autoclaved lake water.

Table 37. M. aeruginosa 2nd culture grown in autoclaved lake water.


200mL BG-11 + 200mL autoclaved lake H2O + 5mL M.a. 8/29/2019 1410 0 0 0.011

200mL BG-11 + 200mL autoclaved lake H2O + 5mL M.a. 9/3/2019 1000 116 4.8 0.058


































100

Table 38. M. aeruginosa grown in reduced media concentration.

Table 39. M. aeruginosa 2nd culture grown in reduced media concentration.

Table 40. Growth of organisms naturally present in lake water.





















50mL lake H2O + 50mL BG-11 8/18/2019 1550 0 0 0.000

50mL lake H2O + 50mL BG-11 9/3/2019 1025 379 15.8 0.060

50mL lake H2O + 50mL BG-11 9/10/2019 0930 167 22.7 0.065

50mL lake H2O + 50mL BG-11 9/17/2019 1045 169 29.8 0.212

50mL lake H2O + 50mL BG-11 9/24/2019 0835 166 36.7 0.280

50mL lake H2O + 50mL BG-11 10/1/2019 0730 167 43.7 0.275

50mL lake H2O + 50mL BG-11 10/8/2019 0745 168 50.7 0.463

50mL lake H2O + 50mL BG-11 10/15/2019 0845 169 57.7 0.625

50mL lake H2O + 50mL BG-11 10/21/2019 0935 145 63.7 0.976

50mL lake H2O + 50mL BG-11 10/29/2019 0800 190 71.7 1.162

50mL lake H2O + 50mL BG-11 11/5/2019 1050 171 78.8 1.345

50mL lake H2O + 50mL BG-11 11/12/2019 0850 166 85.7 1.436

50mL lake H2O + 50mL BG-11 11/19/2019 0930 169 92.7 1.539

50mL lake H2O + 50mL BG-11 11/26/2019 1000 169 99.8 1.537

50mL lake H2O + 50mL BG-11 12/3/2019 0825 166 106.7 0.897

101

Table 41. No growth observed in lake water without growth media.

Table 42. Growth of organisms in lake water with non-autoclaved fertilizer.

Table 43. Growth of R. raciborskii in modified combo media.

Table 44. Growth of R. raciborskii in lake water amended with modified combo media.


Lake Water Only 8/19/2019 1940 0 0 0.000

Lake Water Only 9/17/2019 1125 688 28.6 0.000

Lake Water Only 10/21/2019 1005 815 62.6 0.000

Lake Water Only 10/29/2019 0805 190 70.5 0.000


LW + 1g fertilizer 12/19/2019 1845 0 0 0.0000

LW + 1g fertilizer 12/31/2020 1920 289 12 0.1048

LW + 1g fertilizer 1/28/2020 0940 662 40 0.4096

LW + 1g fertilizer 2/10/2020 1155 314 53 0.2552


50mL Combo + 1.25mL R.r. 12/19/2019 1855 0 0 0.042

50mL Combo + 1.25mL R.r. 12/31/2019 1935 289 12 0.189

50mL Combo + 1.25mL R.r. 1/7/2020 1135 160 19 0.195

50mL Combo + 1.25mL R.r. 1/28/2020 0930 502 40 0.134

50mL Combo + 1.25mL R.r. 2/10/2020 1600 319 53 0.486

50mL Combo + 1.25mL R.r. 2/25/2020 1955 364 68 0.882


50mL Combo + 50mL lake H2O + 2.5mL R.r. 12/19/2019 1900 0 0 0.024






102

Table 45. Growth of R. raciborskii in lake water.

Table 46. Growth of organisms naturally present in lake water with modified combo media.

Table 47. Growth of R. raciborskii in autoclaved lake water amended with modified combo media.

Table 48. R. raciborskii 2nd pure culture.


50mL lake H2O + 1.25mL R.r. 12/19/2019 1850 0 0 0.004

50mL lake H2O + 1.25mL R.r. 12/31/2019 1930 289 12 0.033

50mL lake H2O + 1.25mL R.r. 1/7/2020 1155 160 19 0.057

50mL lake H2O + 1.25mL R.r. 1/28/2020 0955 502 40 0.069

50mL lake H2O + 1.25mL R.r. 2/10/2020 1615 318 53 0.071

50mL lake H2O + 1.25mL R.r. 2/25/2020 2015 364 68 0.071


50mL Combo + 50mL lake H2O 12/19/2019 1905 0 0 0.001

50mL Combo + 50mL lake H2O 1/28/2020 0930 950 40 0.1344


100mL Combo + 100mL ALW + 5mL R.r. 11/8/2019 1510 0 0 0.026






100mL Combo + 2.5mL R.r. 10/22/2019 1000 0 0 0.007

100mL Combo + 2.5mL R.r. 11/5/2019 1120 337 14 0.098

100mL Combo + 2.5mL R.r. 1/7/2020 1150 1513 77 0.396

100mL Combo + 2.5mL R.r. 1/28/2020 1005 502 98 0.4036

100mL Combo + 2.5mL R.r. 2/10/2020 1625 318 111 0.3636

100mL Combo + 2.5mL R.r. 2/25/2020 2010 364 126 0.4016

103

Appendix II – Microcystin Concentrations

Table 49. Lake water toxins

Table 50. Low media concentration toxins.

Date Collected 8/19/2019 19:40 8/19/2019 19:50 9/3/2019 10:25 8/18/2019 16:40

Project Name UNLV Microcystin UNLV Microcystin UNLV Microcystin UNLV Microcystin

Event LC-MS/MS comparison, Round II LC-MS/MS comparison, Round II LC-MS/MS comparison, Round II LC-MS/MS comparison, Round II

Location LW-0 LWBG-0 50LWBG-1 LWBGJuly

Parameter Units

Anatoxin-a ug/L <0.50 <0.50 <0.50 <0.50

Cylindrospermopsin ug/L <0.50 <0.50 <0.50 <0.50

Microcystin-HilR ug/L <0.50 <0.50 <0.50 <0.50

Microcystin-HtyR ug/L <0.50 <0.50 <0.50 <0.50

Microcystin-LA ug/L <0.50 <0.50 <0.50 <0.50

Microcystin-LF ug/L <0.50 <0.50 <0.50 <0.50

Microcystin-LR ug/L <0.50 <0.50 <0.50 <0.50

Microcystin-LW ug/L <0.50 <0.50 <0.50 <0.50

Microcystin-LY ug/L <0.50 <0.50 <0.50 <0.50

Microcystin-RR ug/L <0.50 <0.50 <0.50 <0.50

Microcystin-WR ug/L <0.50 <0.50 <0.50 <0.50

Microcystin-YR ug/L <0.50 <0.50 <0.50 <0.50

Microcystin-dmLR ug/L <0.50 <0.50 <0.50 <0.50

Microcystin-dmRR ug/L <0.50 <0.50 <0.50 <0.50

Nodularin ug/L <0.50 <0.50 <0.50 <0.50

Date Collected 8/19/2019 20:10 8/29/2019 14:45 9/3/2019 10:15

Project Name UNLV Microcystin UNLV Microcystin UNLV Microcystin

Event LC-MS/MS comparison, Round II LC-MS/MS comparison, Round II LC-MS/MS comparison, Round II

Location BG40-0 BG40-1 BG40-2

Parameter Units

Anatoxin-a ug/L <0.50 <0.50 <0.50

Cylindrospermopsin ug/L <0.50 <0.50 <0.50

Microcystin-HilR ug/L <0.50 <0.50 <0.50

Microcystin-HtyR ug/L <0.50 <0.50 <0.50

Microcystin-LA ug/L <0.50 <0.50 <0.50

Microcystin-LF ug/L <0.50 <0.50 <0.50

Microcystin-LR ug/L 2.5 <0.50 <0.50

Microcystin-LW ug/L <0.50 <0.50 <0.50

Microcystin-LY ug/L <0.50 <0.50 <0.50

Microcystin-RR ug/L <0.50 <0.50 <0.50

Microcystin-WR ug/L <0.50 <0.50 <0.50

Microcystin-YR ug/L <0.50 <0.50 <0.50

Microcystin-dmLR ug/L <0.50 <0.50 <0.50

Microcystin-dmRR ug/L <0.50 <0.50 <0.50

Nodularin ug/L <0.50 <0.50 <0.50

104

Table 51. Toxin production in lake water amended with growth media.

Table 52. Toxin production in autoclaved lake water and lake water.

Date Collected 8/18/2019 16:35 8/23/2019 11:20 8/28/2019 18:05 9/3/2019 9:45



Location 200LWBG-0 200LWBG-1 200LWBG-2 200LWBG-3

Parameter Units

Anatoxin-a ug/L <0.50 <0.50 <0.50 <0.50






Microcystin-LR ug/L 1.2 4.5 1.3 4.9








Nodularin ug/L <0.50 <0.50 <0.50 <0.50

Date Collected 8/29/2019 14:10 9/3/2019 10:00 8/18/2019 15:50


Event LC-MS/MS comparison, Round II LC-MS/MS comparison, Round II LC-MS/MS comparison, Round II

Location 200ALWBG-0 200ALWBG-1 50LWBG-0

Parameter Units

Anatoxin-a ug/L <0.50 <0.50 <0.50






Microcystin-LR ug/L 1.4 8.4 <0.50

Microcystin-LW ug/L <0.50 0.78 <0.50





Microcystin-dmLR ug/L <0.50 0.79 <0.50


Nodularin ug/L <0.50 <0.50 <0.50

105

Table 53. Pure culture M. aeruginosa toxin production.

Table 54. Second set of toxin data from M. aeruginosa pure culture.

Date Collected 8/18/2019 16:05 8/23/2019 11:25 8/28/2019 18:10 9/3/2019 9:40



Location 200BG-0 200BG-1 200BG-2 200BG-3

Parameter Units

Anatoxin-a ug/L <0.50 <0.50 <0.50 <0.50


Microcystin-HilR ug/L <0.50 <0.50 0.8 1.3



Microcystin-LF ug/L <0.50 1.5 7.3 12

Microcystin-LR ug/L 3.6 12 43 82

Microcystin-LW ug/L 0.52 1.9 14 24

Microcystin-LY ug/L <0.50 1.4 5.1 8.3




Microcystin-dmLR ug/L 0.55 1.1 3.9 10


Nodularin ug/L <0.50 <0.50 <0.50 <0.50

Date Collected 9/10/2019 9:40 9/17/2019 10:55 9/24/2019 8:45 10/1/2019 7:35


Event LC-MS/MS comparison, Round III LC-MS/MS comparison, Round III LC-MS/MS comparison, Round III LC-MS/MS comparison, Round III

Location 200B-5 200B-6 200B-7 200B-8

Parameter Units

Anatoxin-a ug/L <0.50 <0.50 <0.50 <0.50


Microcystin-HilR ug/L 2.9 3.2 3 3.1



Microcystin-LF ug/L 16 18 18 15

Microcystin-LR ug/L 130 140 130 110

Microcystin-LW ug/L 26 22 2.6 1.4

Microcystin-LY ug/L 8.3 4.8 <0.50 <0.50


Microcystin-WR ug/L 0.53 0.9 1.2 0.92


Microcystin-dmLR ug/L 38 50 41 30


Nodularin ug/L <0.50 <0.50 <0.50 <0.50

106

Table 55. Third set of toxin data from M. aeruginosa pure culture.

Table 56. No toxins detected in low media concentration.

Date Collected 10/8/2019 7:55 10/21/2019 9:40 10/29/2019 6:15


Event LC-MS/MS comparison, Round III LC-MS/MS comparison, Round III LC-MS/MS comparison, Round III

Location 200B-9 200B-10 200B-11

Parameter Units

Anatoxin-a ug/L <0.50 <0.50 <0.50


Microcystin-HilR ug/L 3.7 3.5 4



Microcystin-LF ug/L 17 19 23

Microcystin-LR ug/L 130 160 210

Microcystin-LW ug/L 2.2 2.4 2.7

Microcystin-LY ug/L 0.55 0.69 0.5


Microcystin-WR ug/L 0.95 0.85 0.92


Microcystin-dmLR ug/L 33 29 32


Nodularin ug/L <0.50 <0.50 <0.50

Date Collected 9/10/2019 10:10 9/17/2019 11:15 9/24/2019 9:00 10/1/2019 7:50



Location 40B-4 40B-5 40B-6 40B-7

Parameter Units

Anatoxin-a ug/L <0.50 <0.50 <0.50 <0.50






Microcystin-LR ug/L <0.50 <0.50 <0.50 <0.50








Nodularin ug/L <0.50 <0.50 <0.50 <0.50

107

Table 57. Second set of samples showing no toxin detection in low media concentration.

Table 58. Toxins produced by M. aeruginosa in autoclaved lake water amended with growth media.

Date Collected 10/8/2019 8:10 10/15/2019 9:10 10/21/2019 9:50


Event LC-MS/MS comparison, Round III LC-MS/MS comparison, Round III LC-MS/MS comparison, Round III

Location 40B-8 40B-9 40B-10

Parameter Units

Anatoxin-a ug/L <0.50 <0.50 <0.50






Microcystin-LR ug/L <0.50 <0.50 <0.50

Microcystin-LW ug/L <0.50 <0.50 <0.50





Microcystin-dmLR ug/L <0.50 <0.50 <0.50


Nodularin ug/L <0.50 <0.50 <0.50

Date Collected 9/10/2019 10:15 9/17/2019 11:20 9/24/2019 9:05 10/1/2019 7:55



Location 200ALW-3 200ALW-4 200ALW-5 200ALW-6

Parameter Units

Anatoxin-a ug/L <0.50 <0.50 <0.50 <0.50


Microcystin-HilR ug/L 0.52 1.3 1.3 1.8



Microcystin-LF ug/L 1.7 2.8 3 3.4

Microcystin-LR ug/L 36 130 130 140

Microcystin-LW ug/L 2.8 4 4.4 3.8

Microcystin-LY ug/L 0.86 1.4 1 0.58




Microcystin-dmLR ug/L 6.6 9.9 12 13


Nodularin ug/L <0.50 <0.50 <0.50 <0.50

108

Table 59. High concentrations of toxins produced in M. aeruginosa pure culture.

Table 60. Toxins produced by M. aeruginosa in lake water amended with growth media.

Date Collected 11/5/2019 10:55 11/12/2019 8:55 12/11/2019 12:30

Project Name LC-MS/MS comparison, Round IV LC-MS/MS comparison, Round IV LC-MS/MS comparison, Round IV

Event UNLV Algal toxins, Microcystins UNLV Algal toxins, Microcystins UNLV Algal toxins, Microcystins

Location 200B-13 200B-14 200B-18

Parameter Units

Anatoxin-a ug/L <0.50 <0.50 <0.50


Microcystin-HilR ug/L 9.3 14 15



Microcystin-LF ug/L 47 110 120

Microcystin-LR ug/L 450 970 910

Microcystin-LW ug/L 17 24 8.3

Microcystin-LY ug/L 8.9 21 4.2


Microcystin-WR ug/L 1.4 2 2.6


Microcystin-dmLR ug/L 210 440 470


Nodularin ug/L <0.50 <0.50 <0.50

Date Collected 11/8/2019 18:05 12/3/2019 9:05 1/7/2020 11:25 1/21/2020 6:35

Project Name LC-MS/MS comparison, Round IV LC-MS/MS comparison, Round IV LC-MS/MS comparison, Round IV LC-MS/MS comparison, Round IV

Event UNLV Algal toxins, Microcystins UNLV Algal toxins, Microcystins UNLV Algal toxins, Microcystins UNLV Algal toxins, Microcystins

Location 100BLW-1 100BLW-3 100BLW-7 100BLW-8

Parameter Units

Anatoxin-a ug/L <0.50 <0.50 <0.50 <0.50





Microcystin-LF ug/L <0.50 2.2 2.4 3.5

Microcystin-LR ug/L 1.3 38 40 37

Microcystin-LW ug/L <0.50 3.2 3.4 4.5

Microcystin-LY ug/L <0.50 1.9 1.6 1.6


Microcystin-WR ug/L <0.50 <0.50 <0.50 0.66


Microcystin-dmLR ug/L <0.50 4.7 3.7 3.8


Nodularin ug/L <0.50 <0.50 <0.50 <0.50

109

Table 61. Toxins nearly undetectable in low media concentration.

Table 62. No toxins detected in lake water, and nearly undetectable toxins in low media concentration.

Date Collected 11/26/2019 12:00 12/17/2019 9:30 1/21/2020 5:35 12/31/2019 19:20

Project Name LC-MS/MS comparison, Round IV LC-MS/MS comparison, Round IV LC-MS/MS comparison, Round IV LC-MS/MS comparison, Round IV

Event UNLV Algal toxins, Microcystins UNLV Algal toxins, Microcystins UNLV Algal toxins, Microcystins UNLV Algal toxins, Microcystins

Location 40B-1 40B-3 40B-6 LWF-3

Parameter Units

Anatoxin-a ug/L <0.50 <0.50 <0.50 <0.50






Microcystin-LR ug/L <0.50 0.77 0.7 <0.50








Nodularin ug/L <0.50 <0.50 <0.50 <0.50

Date Collected 3/4/2020 15:06 3/4/2020 15:09 3/4/2020 11:50 3/4/2020 15:00

Project Name Comparison, Round IIV MCs Comparison, Round IIV MCs Comparison, Round IIV MCs Comparison, Round IIV MCs

Event UNLV Algal toxins UNLV Algal toxins UNLV Algal toxins UNLV Algal toxins

Location LWBG-1 LWBG-2 40B-2 40B-3

ParameterUnits

Anatoxin-aug/L <0.50 <0.50 <0.50 <0.50

Cylindrospermopsinug/L <0.50 <0.50 <0.50 <0.50

Microcystin-HilRug/L <0.50 <0.50 <0.50 <0.50

Microcystin-HtyRug/L <0.50 <0.50 <0.50 <0.50

Microcystin-LAug/L <0.50 <0.50 <0.50 <0.50

Microcystin-LFug/L <0.50 <0.50 <0.50 <0.50

Microcystin-LRug/L <0.50 <0.50 0.54 0.59

Microcystin-LWug/L <0.50 <0.50 <0.50 <0.50

Microcystin-LYug/L <0.50 <0.50 <0.50 <0.50

Microcystin-RRug/L <0.50 <0.50 <0.50 <0.50

Microcystin-WRug/L <0.50 <0.50 <0.50 <0.50

Microcystin-YRug/L <0.50 <0.50 <0.50 <0.50

Microcystin-dmLRug/L <0.50 <0.50 <0.50 <0.50

Microcystin-dmRRug/L <0.50 <0.50 <0.50 <0.50

Nodularin ug/L <0.50 <0.50 <0.50 <0.50

110

Table 63. Toxins produced in lake water amended with growth media, and no toxins produced with very low media concentrations.

Date Collected 3/4/2020 11:45 3/4/2020 14:57 3/4/2020 14:30 3/4/2020 14:27

Project Name Comparison, Round IIV MCs Comparison, Round IIV MCs Comparison, Round IIV MCs Comparison, Round IIV MCs

UNLV Algal toxins UNLV Algal toxins UNLV Algal toxins UNLV Algal toxins

100B-1 100B-2 LWP 1BG

Units

ug/L <0.50 <0.50 <0.50 <0.50

ug/L <0.50 <0.50 <0.50 <0.50

ug/L <0.50 <0.50 <0.50 <0.50

ug/L <0.50 <0.50 <0.50 <0.50

ug/L <0.50 <0.50 <0.50 <0.50

ug/L 2.8 3.7 <0.50 <0.50

ug/L 33 29 <0.50 <0.50

ug/L 2.7 3.1 <0.50 <0.50

ug/L 1 0.92 <0.50 <0.50

ug/L <0.50 <0.50 <0.50 <0.50

ug/L 0.7 0.62 <0.50 <0.50

ug/L <0.50 <0.50 <0.50 <0.50

ug/L 1.7 1.3 <0.50 <0.50

ug/L <0.50 <0.50 <0.50 <0.50

ug/L <0.50 <0.50 <0.50 <0.50

111

Appendix III – BMAA Concentrations

Table 64. No BMAA detected in R. raciborskii cultures in media or media + lake water.

Table 65. No BMAA detected in 2nd set of R. raciborskii samples.

Table 66. No BMAA detected in 3rd set of R. raciborskii samples.

Table 67. BMAA detected in cyanobacterial cultures.

Date Collected 12/19/2019 18:55 1/7/2020 11:35 12/19/2019 19:00 12/31/2019 19:40

Project Name Comparison, Round IV BMAA Comparison, Round IV BMAA Comparison, Round IV BMAA Comparison, Round IV BMAA

Event UNLV Algal toxins, BMAA UNLV Algal toxins, BMAA UNLV Algal toxins, BMAA UNLV Algal toxins, BMAA

Location 50C-1 50C-3 50CLW-1 50CLW-2

Parameter Units

beta-Methylamino-L-alanine ug/L <5.0 <5.0 <5.0 <5.0

Date Collected 12/19/2019 18:50 1/7/2020 11:55 1/21/2020 7:25

Project Name Comparison, Round IV BMAA Comparison, Round IV BMAA Comparison, Round IV BMAA

Event UNLV Algal toxins, BMAA UNLV Algal toxins, BMAA UNLV Algal toxins, BMAA

Location 50LW-1 50LW-3 CLW-2

Parameter Units

beta-Methylamino-L-alanine ug/L <5.0 <5.0 <5.0

Date Collected 1/7/2020 11:45 1/21/2020 6:15 11/8/2019 15:10 1/7/2020 11:45



Location 100CALW-2 100CALW-3 100C-1 100C-4

Parameter Units


Date Collected 1/21/2020 5:30 1/21/2020 5:30 1/21/2020 5:30 1/21/2020 5:30



Location MICR SYN NOD ANA

Parameter Units

beta-Methylamino-L-alanine ug/L 30 <5.0 31 49

112

Table 68. BMAA detected in 2nd set of cyanobacterial cultures.

Table 69. No BMAA detected in tap water, bottled water, or lake water, but detected in seafood/cod (SF) and lake water with fertilizer added.

Table 70. Some BMAA detected in R. raciborskii cultures.

Table 71. BMAA detected in another R. raciborskii culture.

Date Collected 1/21/2020 5:30 1/21/2020 5:30 1/21/2020 5:30 1/21/2020 5:30



Location OSC LYN NOS APH

Parameter Units

beta-Methylamino-L-alanine ug/L 6.5 230 20 <5.0

Date Collected 1/21/2020 5:25 1/21/2020 5:25 1/21/2020 7:35 1/21/2020 5:25 1/21/2020 6:50

Project Name Comparison, Round IV BMAA Comparison, Round IV BMAA Comparison, Round IV BMAA Comparison, Round IV BMAA Comparison, Round IV BMAA

Event UNLV Algal toxins, BMAA UNLV Algal toxins, BMAA UNLV Algal toxins, BMAA UNLV Algal toxins, BMAA UNLV Algal toxins, BMAA

Location TW BW SF LW LWF

Parameter Units

beta-Methylamino-L-alanine ug/L <5.0 <5.0 >500 <5.0 25

Date Collected 1/21/2020 6:20 1/21/2020 6:25 1/21/2020 6:30 1/21/2020 7:25

Project Name Comparison, Round IV BMAA/Mic. Comparison, Round IV BMAA/Mic. Comparison, Round IV BMAA/Mic. Comparison, Round IV BMAA/Mic.


Location 50C 50CLW 50LW CLW

ParameterUnits

Cylindrospermopsinug/L

beta-Methylamino-L-alanineug/L <5.0 6.1 <5.0 5.5

Date Collected 3/4/2020 14:18 3/4/2020 14:16 3/4/2020 14:13 3/4/2020 14:10

Project Name Comparison, Round IIV BMAA Comparison, Round IIV BMAA Comparison, Round IIV BMAA Comparison, Round IIV BMAA


Location 50C 50CL 50L CL

Parameter Units

beta-Methylamino-L-alanine ug/L 5 <5.0 <5.0 <5.0

Date Collected 3/4/2020 14:03 3/4/2020 14:03 3/4/2020 14:02 3/4/2020 14:02



Location MICR SYN NOD ANA

Parameter Units

beta-Methylamino-L-alanine ug/L 15 <5.0 <5.0 <5.0

Date Collected 3/4/2020 14:02 3/4/2020 14:01 3/4/2020 14:01 3/4/2020 14:03



Location OSC LYN NOS APH

Parameter Units

beta-Methylamino-L-alanine ug/L <5.0 220 16 8.1

113

Table 72. No BMAA detected in a 2nd set of tap water, bottled water, lake water, and DI water samples.

Table 73. BMAA detected in seafood samples.

Table 74. BMAA detected in 2nd set of seafood samples.

Date Collected 3/4/2020 14:00 3/4/2020 14:00 3/4/2020 14:00 3/4/2020 15:27



Location TW BW LW SF-1

Parameter Units


Date Collected 3/4/2020 15:30 3/4/2020 15:33 3/4/2020 15:36

Project Name Comparison, Round IIV BMAA Comparison, Round IIV BMAA Comparison, Round IIV BMAA

Event UNLV Algal toxins UNLV Algal toxins UNLV Algal toxins

Location SF-2 SF-3 SF-4

Parameter Units

beta-Methylamino-L-alanine ug/L 770 21 55

Date Collected 3/4/2020 15:39 3/4/2020 15:42 3/4/2020 15:45

Project Name Comparison, Round IIV BMAA Comparison, Round IIV BMAA Comparison, Round IIV BMAA

Event UNLV Algal toxins UNLV Algal toxins UNLV Algal toxins

Location SF-5 SF-6 SF-7

Parameter Units

beta-Methylamino-L-alanine ug/L 110 490 12

114

Appendix IV – QPCR Results

Table 75. Near-limit of detection of mcyE genes.

Table 76. Detection of mcyE genes from surface growth accumulation.

Run DateTime 4/1/2020 4:53

Operator Tim

ThermalCycleParams

Sample Information

Well Sample Name Detector NameReporter Task Ct

25 MC+ mcyE FAM Unknown 39.798115

26 MC+ mcyE FAM Unknown 39.208076

27 MC- mcyE FAM Unknown Undetermined

28 MC- mcyE FAM Unknown Undetermined

1 NTC mcyE FAM NTC Undetermined


59 PC mcyE FAM Unknown 25.281399


Run DateTime 3/31/2020 23:30

Operator Tim

ThermalCycleParams

Sample Information






25 Sample mcyE FAM Unknown 23.625135

26 Sample mcyE FAM Unknown 23.895535

115

Table 77. Cell count standard curve, freeze/thaw method comparison, and blind experiment mcyE qPCR results.

Run DateTime 3/22/2020 10:19

Operator Tim

ThermalCycleParams

Sample Information


35 -1CC mcyE FAM Unknown 30.55693




33 0CC mcyE FAM Unknown 26.412296

34 0CC mcyE FAM Unknown 26.313282

31 10XCC mcyE FAM Unknown 24.640675

32 10XCC mcyE FAM Unknown 24.4024

43 10XExtr mcyE FAM Unknown 37.918484

44 10XExtr mcyE FAM Unknown 38.042103

41 10XHS mcyE FAM Unknown 28.480225

42 10XHS mcyE FAM Unknown 33.04731

25 A blind mcyE FAM Unknown Undetermined


27 B blind mcyE FAM Unknown Undetermined


29 C blind mcyE FAM Unknown 33.17194


47 Extr mcyE FAM Unknown 36.778748

48 Extr mcyE FAM Unknown 37.10371

45 HS mcyE FAM Unknown 39.84684


71 MAT10X mcyE FAM Unknown 25.089241




39 SD mcyE FAM Unknown Undetermined

40 SD mcyE FAM Unknown Undetermined

116

Table 78. Freeze/thaw, blind experiment, and cell count dilution mcyE qPCR results.

Run DateTime 3/13/2020 23:18

Operator Tim

ThermalCycleParams

Sample Information


33 100HS mcyE FAM Unknown Undetermined

34 100HS mcyE FAM Unknown Undetermined

31 100HS10X mcyE FAM Unknown 31.499954

32 100HS10X mcyE FAM Unknown 30.850222



37 ALWHS mcyE FAM Unknown 30.324953

38 ALWHS mcyE FAM Unknown 30.301079

35 ALWHS10X mcyE FAM Unknown 28.718912

36 ALWHS10X mcyE FAM Unknown 28.772165





49 CC-1 mcyE FAM Unknown Undetermined




47 CC0 mcyE FAM Unknown Undetermined

48 CC0 mcyE FAM Unknown 35.587013

45 CC10X mcyE FAM Unknown 26.3922

46 CC10X mcyE FAM Unknown 24.6983

39 LWBG mcyE FAM Unknown Undetermined




41 MaExtr mcyE FAM Unknown 28.532063

42 MaExtr mcyE FAM Unknown 28.457579

43 MaHS mcyE FAM Unknown 22.095633

44 MaHS mcyE FAM Unknown 22.21622



117

Table 79. Pure culture M. aeruginosa, reduced media concentration, and DNA extraction technique comparison mcyE gene qPCR.

Run DateTime 2/11/2020 12:07

Operator Tim

ThermalCycleParams

Sample Information


25 200-1 mcyE FAM Unknown 35.596695

26 200-1 mcyE FAM Unknown Undetermined

29 200-10 mcyE FAM Unknown 26.964643

30 200-10 mcyE FAM Unknown 28.369709









1 A1 mcyE FAM NTC Undetermined

2 A2 mcyE FAM NTC Undetermined

59 Cdiff-3 mcyE FAM Unknown Undetermined

60 Cdiff-3 mcyE FAM Unknown Undetermined

39 EXTR mcyE FAM Unknown 27.76922

40 EXTR mcyE FAM Unknown 27.935762





43 MAT mcyE FAM Unknown 27.080553

44 MAT mcyE FAM Unknown 25.335548

118

Appendix V - Growth Media

Bold 3N Media Recipe

1. NaNO3 (Fisher BP360-500), 30 mL/L, 10 g/400mL dH2O, 8.82 mM

2. CaCl2·2H O (Sigma C-3881), 10 mL/L, 1 g/400mL dH2O, 0.17 mM

3. MgSO4·7H O (Sigma 230391), 10 mL/L, 3 g/400mL dH2O, 0.3 mM

4. K2HPO4 (Sigma P 3786), 10 mL/L, 3 g/400mL dH2O, 0.43 mM

5. KH2PO4 (Sigma P 0662), 10 mL/L, 7 g/400mL dH2O, 1.29 mM

6. NaCl (Fisher S271-500), 10 mL/L, 1 g/400mL dH2O, 0.43 mM

7. P-IV Metal Solution 6 mL/L

a. Na2EDTA•2H2O (Sigma ED255), 0.75 g/L, 2 mM

b. FeCl3•6H2O (Sigma F-1513), 0.097 g/L, 0.36 mM

c. MnCl2•4H2O (Baker 2540), 0.041 g/L, 0.21 mM

d. ZnCl2 (Sigma Z-0152), 0.005 g/L, 0.037 mM

e. CoCl2•6H2O (Sigma C-3169), 0.002 g/L, 0.0084 mM

f. Na2MoO4•2H2O (J.T. Baker 3764), 0.004 g/L, 0.017 mM

8 Soilwater: GR+ Medium 40 mL/L

a. Green House Soil, 1 tsp

b. CaCO3(optional) (Fisher C 64), 1 mg, 0.05 mM

c. dH2O, 200 mL

9 Vitamin B12, 1 mL/L

Modified Bold 3N Media Recipe (Bold 3N with the following additional solutions):

1. Biotin Vitamin Solution 1 mL/L

a. HEPES buffer pH 7.8 (Sigma H-3375) 2.4 g/200 mL dH2O

b. Biotin (Sigma B-4639) 0.005 g/200 mL dH2O

2. Thiamine Vitamin Solution1 mL/L

a. HEPES buffer pH 7.8 (Sigma H-3375) 2.4 g/200 mL dH2O

b. Thiamine (Sigma T-1270) 0.067 g/200 mL dH2O

119

BG-11 Media Recipe

1 NaNO3 (Fisher BP360-500) 10 mL/L 30 g/200 mL dH2O 17.6 mM

2 K2HPO4 (Sigma P 3786) 10 mL/L 0.8 g/200 mL dH2O 0.23 mM

3 MgSO4•7H2O (Sigma 230391) 10 mL/L 1.5 g/200 mL dH2O 0.3 mM

4 CaCl2•2H2O (Sigma C-3881) 10 mL/L 0.72 g/200 mL dH2O 0.24 mM

5 Citric Acid•H2O (Fisher A 104) 10 mL/L 0.12 g/200 mL dH2O 0.031 mM

6 Ferric Ammonium Citrate 10 mL/L 0.12 g/200 mL dH2O 0.021 mM

7 Na2EDTA•2H2O (Sigma ED255) 10 mL/L 0.02 g/200 mL dH2O 0.0027 mM

8 Na2CO3 (Baker 3604) 10 mL/L 0.4 g/200 mL dH2O 0.19 mM

9 BG-11 Trace Metals Solution 1 mL/L

a. H3BO3 (Baker 0084) 2.86 g/L 46 mM

b. MnCl2•4H2O (Baker 2540) 1.81 g/L 9 mM

c. ZnSO4•7H2O (Sigma Z 0251) 0.22 g/L 0.77 mM

d. Na2MoO4•2H2O (J.T. Baker 3764) 0.39 g/L 1.6 mM

e. CuSO4•5H2O (MCIB 3M11) 0.079 g/L 0.3 mM

f. Co(NO3)2•6H2O (ACROS 10026-22-9) 49.4 mg/L 0.17 mM

10 Sodium Thiosulfate Pentahydrate (agar media only,sterile) (Baker 3946) 1mL/L 49.8 g/200 mL dH2O 1 mM

Enriched Seawater Medium Recipe

1 Pasteurized Seawater 1 L

2 Enrichment Solution for Seawater Medium 20 mL/L

a. NaNO3 (Fisher BP360-500) 4.7 g/2 L 27.65 mM

b. Na2glycerophosphate•5H2O (Sigma G 6501 ) 0.7 g/2 L 1.6 mM

c. ES Fe Solution 325 mL/2 L

i. Fe(NH4)2(SO4)2•6H2O (Sigma F-1513) 1.4 g/2 L 1.8 mM

ii. Na2EDTA•2H2O (Sigma ED255) 1.2 g/2 L 1.6 mM

d. P-II Metal Solution 325 mL/2 L

i. Na2EDTA•2H2O (Sigma ED255) 0.1 g/100 mL 2.7 mM

120

ii. H3BO3 (Baker 0084) 0.114 g/100 mL 18.4 mM

iii. FeCl3•6H2O (Sigma F-1513) 4.9 mg/100 mL 0.18 mM

iv. MnSO4•H2O (Sigma M8179) 16.4 mg/100 mL 0.97 mM

v. ZnSO4•7H2O (Sigma Z 0251) 2.2 mg/100 mL 0.07 mM

vi. CoCl2•6H2O (Sigma C-3169) 0.48 mg/100 mL 0.02 mM

e. HEPES buffer (Sigma H-3375) 6.5 g/2 L 14 mM

f. Vitamin B12 3 mL/2 L

i. HEPES buffer pH 7.8 (Sigma H-3375) 2.4 g/200 mL dH2O

ii. Vitamin B12 (cyanocobalamin, (Sigma V-6629)) 0.027 g/200 mL dH2

g. Biotin Vitamin Solution 3 mL/2 L


ii. Biotin (Sigma B-4639) 0.005 g/200 mL dH2

h. Thiamine Vitamin Solution 3 mL/2 L


ii. Thiamine (Sigma T-1270) 0.067 g/200 mL dH2O

BG-11(-N) Media Recipe

1 K2HPO4 (Sigma P 3786) 10 mL/L 0.8 g/200 mL 0.22 mM

2 MgSO4•7H2O (Sigma 230391) 10 mL/L 1.5 g/200 mL 0.3 mM

3 CaCl2•2H2O (Sigma C-3881) 10 mL/L 0.72 g/200 mL 0.24 mM

4 Citric Acid•H2O (Fisher A 104) 10 mL/L 0.12 g/200 mL 0.012 mM

5 Ferric Ammonium Citrate 10 mL/L 0.12 g/200 mL 0.02 mM

6 Na2EDTA•2H2O (Sigma ED255) 10 mL/L 0.02 g/200 mL 0.002 mM

7 Na2CO3 (Baker 3604) 10 mL/L 0.4 g/200 mL 0.18 mM

8 BG-11 Trace Metals Solution 1 mL/L

a. H3BO3 (Baker 0084) 2.86 g/L 46 mM

b. MnCl2•4H2O (Baker 2540) 1.81 g/L 9 mM

c. ZnSO4•7H2O (Sigma Z 0251) 0.22 g/L 0.77 mM

121

d. Na2MoO4•2H2O (J.T. Baker 3764) 0.39 g/L 1.6 mM

e. CuSO4•5H2O (MCIB 3M11) 0.079 g/L 0.3 mM

f. Co(NO3)2•6H2O (ACROS 10026-22-9) 49.4 mg/L 0.17 mM

9 Sodium Thiosulfate Pentahydrate (agar media only,sterile) (Baker 3946) 1 mL/L 49.6 g/200 mL 1 mM

AF6 Media Recipe

1 MES buffer 400 mg 2.05 x 10-3 M

2 NaNO3 140 g L-1 dH2O 1 mL 1.65 x 10-3 M

3 NH4NO3 22 g L-1 dH2O 1 mL 2.75 x 10-4 M

4 MgSO4 7H2O 30 g L-1 dH2O 1 mL 1.22 x 10-5 M

5 K2HPO4 5 g L-1 dH2O 1 mL 2.87 x 10-5 M

6 KH2PO4 10 g L-1 dH2O 1 mL 7.35 x 10-5 M

7 CaCl2 2H2O 10 g L-1 dH2O 1 mL 6.80 x 10-5 M

8 Fe-citrate 2 g L-1 dH2O 1 mL 8.17 x 10-6 M

9 Citric acid 2 g L-1 dH2O 1 mL 1.04 x 10-5 M

10 Trace metals solution 1 mL

a Na2EDTA 2H2O --- 5.000 g 1.34 x 10-5 M

b FeCl3 6H2O --- 0.98 g 3.63 x 10-6M

c MnCl2 4H2O --- 0.18 g 9.10 x 10-7 M

d ZnSO4 7H2O --- 0.11 g 3.83 x 10-7 M

e CoCl2 6H2O 20.0 g L-1 dH2O 1 mL 8.41 x 10-8M

f Na2MoO4 2H2O 12.5 g L-1 dH2O 1 mL 5.17 x 10-8 M

11 Vitamin solution 1 mL

a thiamine (vit. B1) --- 10 mg 2.96 x 10-8 M

b biotin (vit. H) 2.0 g L-1 dH2O 1 mL 8.19 x 10-9 M

c cyanocobalomin (vit. B12) 1.0 g L-1 dH2O 1 mL 7.38 x 10-10 M

d pyridoxine (vit. B6) 1.0 g L-1 dH2O 1 mL 5.91 x 10-9 M

122

DYV-Medium (DYV-M) Media Recipe

1 MES --- 200 mg 1.02 x 10-3 M

2 MgSO4 • 7H2O 50 g/L dH2O 1 mL 2.03 x 10-4 M

3 KCl 3 g/L dH2O 1 mL 4.02 x 10-5 M

4 NH4Cl 2.68 g/L dH2O 1 mL 5.01 x 10-5 M

5 NaNO3 20 g/L dH2O 1 mL 2.35 x 10-4 M

6 Na2 b-glycerophosphate 2.16 g/L dH2O 1 mL 1.00 x 10-5 M

7 H3BO3 0.8 g/L dH2O 1 mL 1.29 x 10-5 M

8 Na2SiO3 • 9 H2O 14 g/L dH2O 1 mL 4.93 x 10-5 M

9 CaCl2 • 2 H2O 75 g/L dH2O 1 mL 6.76 x 10-4 M

10 Trace element solution 1 mL

a Na2EDTA • 2H2O --- 8.0 g 2.15 x 10-5 M

b FeCl3 • 6 H2O --- 1.0 g 3.70 x 10-6 M

c MnCl2 • 4H2O --- 200 mg 1.01 x 10-6 M

d ZnSO4 • 7H2O --- 40 mg 1.39 x 10-7 M

e CoCl2 • 6H2O 8.0 g L-1 dH2O 1 mL 3.36 x 10-8 M

f Na2MoO4 • 2H2O 20.0 g L-1 dH2O 1 mL 8.27 x 10-8 M

g Na3VO4 • 10H2O 2.0 g L-1 dH2O 1 mL 5.49 x 10-9 M

h H2SeO3 4 g L-1 dH2O 1 mL 2.31 x 10-8 M

11 f/2 vitamin solution 0.5 mL

a thiamine · HCl (vit. B1) --- 200 mg 2.96 x 10-7 M

b biotin (vit. H) 0.1 g/L dH2O 10mL 2.05 x 10-9 M

c cyanocobalamin (vit. B12) 1.0 g/L dH2O 1 mL 3.69 x 10-10 M

Black Sea Media Recipe

To prepare, add 500 mL of f/2 medium to 500 mL of de-ionized water. Autoclave.

f/2 media recipe:

1 NaNO3 75 g/L dH2O 1 mL 8.82 x 10-4 M

123

2 NaH2PO4 H2O 5 g/L dH2O 1 mL 3.62 x 10-5 M

3 Na2SiO3 9H2O 30 g/L dH2O 1 mL 1.06 x 10-4 M

4 trace metal solution 1 mL

a FeCl3 6H2O --- 3.15 g 1.17 x 10-5 M

b Na2EDTA 2H2O --- 4.36 g 1.17 x 10-5 M

c CuSO4 5H2O 9.8 g/L dH2O 1 mL 3.93 x 10-8 M

d Na2MoO4 2H2O 6.3 g/L dH2O 1 mL 2.60 x 10-8 M

e ZnSO4 7H2O 22.0 g/L dH2O 1 mL 7.65 x 10-8 M

f CoCl2 6H2O 10.0 g/L dH2O 1 mL 4.20 x 10-8 M

g MnCl2 4H2O 180.0 g/L dH2O 1 mL 9.10 x 10-7 M

5 vitamin solution 0.5 mL

a thiamine HCl (vit. B1) --- 200 mg 2.96 x 10-7 M

b biotin (vit. H) 0.1 g/L dH2O 10 mL 2.05 x 10-9 M


YBC-II Media Recipe

Weigh out and add the following to 900mL of high quality dH2O:

1 NaCl 24.55 4.20X10-1

2 KCl 0.75 1.00X10-2

3 NaHCO3 0.21 2.50X10-3

4 H3BO3 0.036 5.80X10-4

5 KBr 0.1157 9.72X10-4

6 MgCl2· 6H2O 4.07 2.00X10-2

7 CaCl2·2H2O 1.47 1.00X10-2

8 MgSO4·7H2O 6.18 2.50X10-2

Make the following primary (1°) stock solutions and add the recommended amounts to the above 900mL of high quality dH2O:

1 NaF 2.94/liter 7.00X10-2 1mL 7.00X10-5

2 SrCl2· 6H2O 17.4g/liter 6.50X10-2 1mL 6.50X10-5

124

3 KH2PO4 6.8g/liter 5.00X10-2 1mL 5.00X10-5

4 Na2EDTA 0.74g/liter 2.00X10-3 1mL 2.00X10-6

5 FeCl3· 6H2O 0.11g/liter 4.07X10-4 1mL 4.07X10-7

6 MnCl2· 4H2O 0.04g/liter 2.00X10-4 100µL 2.00X10-8

7 ZnSO4· 7H2O 0.012g/liter 4.00X10-5 100µL 4.00X10-9

8 Na2MoO4· 2H2O 0.027g/liter 1.1X10-4 100µL 1.10X10-8

9 CoCl2· 6H2O 0.06g/liter 2.5X10-4 10µL 2.50X10-9

10 CuSO4· 5H2O 0.025g/liter 1.00X10-4 10µL 1.00X10-9

11 f/2 vitamins 1.2mL

a thiamine HCl (vit. B1) --- 200 mg 2.96 x 10-7 M

b biotin (vit. H) 0.1 g/L dH2O 10 mL 2.05 x 10-9 M


COMBO Media Recipe

NaNO3 85.01 g L-1 dH2O 1 mL 1.00x10-3 M

CaCl2 2H2O 36.76 g L-1 dH2O 1 mL 2.50x10-4 M

MgSO4 7H2O 36.97 g L-1 dH2O 1 mL 1.66x10-4 M

NaHCO3 12.60 g L-1 dH2O 1 mL 1.50x10-4 M

Na2SiO3 9H2O 28.42 g L-1 dH2O 1 mL 1.00x10-4 M

K2HPO4 8.71 g L-1 dH2O 1 mL 5.00x10-5 M

H3BO3 1.0 g L-1 dH2O 1 mL 1.62x10-5 M

KCl 7.45 g L-1 dH2O 1 mL 1.00x10-4 M

Algal trace elements solution 1 mL

Na2EDTA2H2O --- 4.36 g 1.17x10-5 M

FeCl3 6H2O --- 1.00 g 3.70x10-6 M

CuSO4 5H2O 10.0 gL-1 dH2O 1 mL 4.01x10-9 M

ZnSO4 7H2O 22.0 g L-1 dH2O 1 mL 7.65x10-8 M

125

CoCl2 6H2O 10.0 g L-1 dH2O 1 mL 4.20x10-8 M

MnCl2 4H2O 180.0 g L-1 dH2O 1 mL 9.10x10-7 M

Na2MoO4 2H2O 6.0 g L-1 dH2O 1 mL 2.48x10-8 M

H2SeO3 1.6 g L-1 dH2O 1 mL 1.24x10-8 M

Na3VO4 1.8 g L-1 dH2O 1 mL 9.79x10-9 M

Animal trace elements solution 1 mL

LiCl 0.31 g L-1 dH2O 1 mL7.31x10-6 M

RbCl 0.07 g L-1 dH2O 1 mL5.79x10-7 M

SrCl2 6H2O 0.15 g L-1 dH2O 1 mL5.63x10-7 M

NaBr 0.016 g L-1 dH2O 1 mL1.55x10-7 M

KI 0.0033 g L-1 dH2O 1 mL1.99x10-8 M

Vitamin solution 1 mL

thiamine · HCl (vit. B1) --- 100 mg 2.96x10-7 M

biotin (vit. H) 0.5 g/L dH2O 1 mL 2.05x10-9 M

cyanocobalamin (vit. B12) 0.55 g/L dH2O 1 mL 4.06x10-10 M

Jaworski Media Recipe (added per 200 mL):

(1) Ca(NO3)2.4H2O 4.0 g

(2) KH2PO4 2.48 g

(3) MgSO4.7H2O 10.0 g

(4) NaHCO3 3.18 g

(5) EDTAFeNa 0.45 g

EDTANa2 0.45 g

(6) H3BO3 0.496 g

MnCl2.4H2O 0.278 g

(NH4)6Mo7O24.4H2O 0.20 g

(7) Cyanocobalamin 0.008 g

Thiamine HCl 0.008 g

126

Biotin 0.008 g

(8) NaNO3 16.0 g

(9) Na2HPO4.12H2O 7.2 g

127

Appendix VI – Culture Images

Figure 25. Standard 200 mL BG-11 + 5 mL M. aeruginosa Kützing pure culture.

Figure 26. Typical 200 mL BG-11 + 200 mL autoclaved lake water + 5 mL M. aeruginosa culture.

128

Figure 27. 100 mL Lake Water + 100 mL BG-11 + 5 mL M. aeruginosa.

Figure 28. 200 mL Lake Water + 40 mL BG-11 + 5 mL M. aeruginosa.

129

Figure 29. 100 mL Lake Water + 40 mL BG-11 + 5 mL M. aeruginosa culture results in growth that is not buoyant and settles to the bottom of the flask. The green color at the surface of the water is an optical illusion, reflected from the growth below.

Figure 30. 50 mL lake water + 50 mL BG-11 media, no Microcystis added.

130

Figure 31. 400x photo of cells naturally present in Lake Mead water and cultivated using BG-11 media.

Figure 32. Cells grown from organisms naturally present in Lake Mead water are not buoyant.

131

Figure 33. No growth in autoclaved lake water.

Figure 34. No growth in autoclaved lake water amended with BG-11.

132

Figure 35. No growth in lake water with no media added.

Figure 36. M. aeruginosa grows in autoclaved lake water.

133

Figure 37. M. aeruginosa grows at very low media concentration (1 mL BG-11).

Figure 38. Fertilizer used to mimic a source of stormwater pollution in Lake Mead.

134

Figure 39. Nitrogen and phosphorus composition of the fertilizer used in this study.

Figure 40. 1 g of non-autoclaved fertilizer promoted growth in Lake Mead water.

135

Figure 41. The fertilizer in the left flask was not autoclaved prior to adding it to Lake Mead water. The fertilizer in the right flask was autoclaved, but it solidified, and digging it out damaged the fertilizer pellets. The fertilizer in the center flask was autoclaved, and did not produce growth.

Figure 42. R. raciborskii pure cultures turn orange over time.

136

Figure 43. R. raciborskii culture is green before turning orange over time.

Figure 44. Color difference between lake water cultured in Combo and BG-11 media.

137

References

Agudo A., Canto K.P., Chan P.C., Chorus I., Falconer I.R., and Fan A. 2010. IARC monographs on the evaluation of carcinogenic risks to humans Ingested nitrate and nitrite, and cyanobacterial peptide toxins. IARC Monogr. Eval. Carcinog. Risks Hum. 94:1-412.

Ajjawi I., Verruto J., Aqui M., Soriaga L.B., Coppersmith J., Kwok K., Peach L., Orchard E., Kalb R., Xu W., Carlson T.J., Francis K., Konigsfeld K., Bartalis J., Schultz A., Lambert W., Schwartz A.S., Brown R., and Moellering E.R. 2017. Lipid production in Nannochloropsis gaditana is doubled by decreasing expression of a single transcriptional regulator. Nat Biotechnol. 35:647–652.

Allen J.I., Smyth T.J., Siddorn J.R., and Holt M. 2008. How well can we forecast high biomass algal bloom events in a eutrophic coastal sea? Harmful Algae. 8:70–76.

Arnold A., Edgren D.C., and Palladino V.S. 1953. Amyotrophic lateral sclerosis; fifty cases observed on Guam. J Nerv Ment Dis. 117:135–139.

Baker J.A., Neilan B.A., Entsch B., and McKay D.B. 2001. Identification of cyanobacteria and their toxigenicity in environmental samples by rapid molecular analysis. Environ. Toxicol, 16 (6):472-482.

Balbus J.M., Boxall A.B.A., Fenske R.A., McKone T.E., and Zeise L. 2013. Implications of global climate change for the assessment and management of human health risks of chemicals in the natural environment. Environmental Toxicology and Chemistry. 32(1):62-78.

Beaver J.R., Kirsch J.E., Tausz C.E., Samples E.E., Renicker T.R., Scotese K.C., McMaster H.A., Blasius-Wert B.J., Zimba P.V., and Casamatta D.A. 2018. Long-term trends in seasonal plankton dynamics in Lake Mead (Nevada-Arizona, USA) and implications for climate change. Hydrobiologia.

Beversdorf L.J., Miller T.R., and McMahon K.D. 2015.Long-term monitoring reveals carbon-nitrogen metabolism key to microcystin production in eutrophic lakes. Frontiers in Microbiology. 6(456).

Bouaicha N., Miles C.O., Beach D.G., Labidi Z., Djabri A., Benayache N.Y., and Nguyen-Quang T. 2019. Structural Diversity, Characterization and Toxicology of Microcystins. Toxins. 11(12):714.

Borgono C.A. and Diamandis E.P. 2004. The emerging roles of human tissue kallikreins in cancer. Nature Reviews. Cancer. pp. 4876–890.

Bortoli S., Oliveira-Silva D., Kruger T., Dorr F.A., Colepicolo P., Volmer D.A., and Pinto E. 2014. Growth and microcystin production of a Brazilian Microcystis aeruginosa strain (LTPNA 02) under different nutrient conditions. Revista Brasileira de Farmacognosia. 24:389-398.

138

Brand L.E. 2009. Human exposure to cyanobacteria and BMAA. Amyotrophic Lateral Sclerosis. (Supplement 2): 85-95.

Brand L.E., Pablo J., Compton A., Hammerschlag N., and Mash D.C. 2010. Cyanobacterial Blooms and Occurrence of the neurotoxin beta-N-methylamino-L-alanine (BMAA) in South Florida Aquatic Food Webs. Harmful Algae. 9(6):620-635.

Briand E., Escoffier N., Straub C., Sabart M., Quiblier C., and Humbert J.F. 2009. Spatiotemporal changes in the genetic diversity of a bloom-forming Microcystis aeruginosa (cyanobacteria) population. ISME J. 3: 419–429.

Callieri C., Cronberg G., and Stockner J. 2012. Freshwater Picocyanobacteria: Single Cells, Microcolonies and Colonial Forms. In: Ecology of Cyanobacteria: Their Diversity in Time and Space, 2nd ed. pp 229-269.

Campo E., Lezcano M.A., Agha R., Cires S., Quesada A., and El-Shehawy R. 2013. First TaqMan Assay to Identify and Quantify the Cylindrospermopsin-Producing Cyanobacterium Aphanizomenon ovalisporum in Water. Advances in Microbiology. (3), 430-437.

Carmichael W.W., Biggs D.F., and Gorham P.R., 1975. Toxicology and pharmacological action of Anabaena flos-aquae toxin. Science 187, 542–544.

Carmichael W.W., and Gorham P.R. 1978. Anatoxins from clones of Anabaena flos-aquae isolated from lakes of Western Canada Mitt. int. Verein. Limnol., 21:285. Carmichael W.W. 2001. Health effects of toxin producing cyanobacteria: ‘‘the CyanoHABS’’. Hum. Ecol. Risk Assess. 7 (5), 1393–1407.

Chen L., Zhang X., Zhou W., Qiao Q., Liang H., Li G., Wang J., and Cai F. 2013. The Interactive Effects of Cytoskeleton Disruption and Mitochondria Dysfunction Lead to Reproductive Toxicity Induced by Microcystin-LR. PLOS ONE. 8(1).

Chernova, E., Sidelev S., Russkikh I., Voyakina E., and Zhakovskaya Z. 2018. First observation of microcystin- and anatoxin-a-producing cyanobacteria in the easternmost part of the Gulf of Finland (the Baltic Sea). Toxicon. 157:18-24.

Chlipala G., and Orjala J. 2011. Chemodiversity in Freshwater and Terrestrial Cyanobacteria – A Source for Drug Discovery. Current drug targets. 12. 1654-73.

Chorus I., and Bartram J. 1999 Toxic Cyanobacteria in Water—A Guide to Their Public Health Consequences, Monitoring and Management. Routledge, London and New York.

Cox, P., and Sacks O. 2002. Cycad neurotoxins, consumption of flying foxes, and ALS-PDC disease in Guam. Neurology. 58. 956-9.

Cox P., Banack S., Murch S., Rasmussen U., Tien G., Bidigare R., Metcalf J., Morrison L., Codd G., and Bergman B. 2005. Diverse taxa of cyanobacteria produce β-N-methylamino-L-alanine, a neurotoxic amino acid. PNAS. 102(14): 5074-5078.

139

Crosby L.D., and Criddle C.S. 2003. Understanding bias in microbial community analysis techniques due to rrn operon copy number heterogeneity. BioTechniques. 34:790-803.

Davis T.W., Berry D.L., Boyer G.L., and Gobler C.J. 2009. The effects of temperature and nutrients on the growth and dynamics of toxic and non-toxic strains of Microcystis during cyanobacteria blooms. Harmful Algae. 8(5):715-725.

de Figueiredo D.R., Azeiteiro U.M., Esteves S.M., Goncalves F.J.M., and Pereira M.J. 2004. Microcystin-producing blooms – a serious global public health issue. Ecotoxicology and Environmental Safety. 59:151-163.

Devlin J.P., Edwards O.E., Gorham P.R., Hunter N.R., Pike R.K., and Stavric B. 1977. Anatoxin-a, a toxic alkaloid from Anabaena-flos-aquae NRC-44h. Can. J. Chem., 55:1367-1371.

Dittmann E., Neilan B.A., Erhard M., and von Döhren H. & Börner T. 1997. Insertional mutagenesis of a peptide synthetase gene that is responsible for hepatotoxin production in the cyanobacterium Microcystis aeruginosa PCC 7806. Mol Microbiol 26: 779–787.

Dodds W.K., Bouska W.W., Eitzmann J.L., Pilger T.J., Pitts K.L., Riley A.J., Schloesser J.T., and Thornbrugh D.J. 2009. Eutrophication of U.S. Freshwaters: Analysis of Potential Economic Damages. Environmental Science and Technology. 43(1):12-19.

Downing J.A., Watson S.B., and McCauley E. 2001. Predicting Cyanobacteria dominance in lakes. Can. J. Fish. Aquat. Sci. 58: 1905-1908.

Downing S., Banack S.A., Metcalf J.S., Cox P.A., and Downing T.G. 2011. Nitrogen starvation of cyanobacteria results in the production of β-N-methylamino-L-alanine. Toxicon. 58:187-194.

Downing S., Scott L., Zguna N., and Downing T. 2018. Human Scalp Hair as an Indicator of Exposure to the Environmental Toxin β-N-methylamino-L-alanine. Toxins. 10(14).

Drobac D., Tokodi N., Lujic J., Marinovic Z., Subakov-Simic G., Dulic T., Vazic T., Nybom S., Meriluoto J., Codd G.A., and Svircev Z. 2016. Cyanobacteria and cyanotoxins in fishponds and their effects on fish tissue. Harmful Algae. 55:66-76.

Duncan M.W., Steele J.C., Kopin I.J., and Markey S.P. 1990. 2-Amino-3-(methylamino)-propanoic acid (BMAA) in cycad flour: an unlikely cause of amyotrophic lateral sclerosis and parkinsonism-dementia of Guam. Neurology 40: 767-772.

Duarte J.H., and Costa J.A.V. 2018. Blue light emitting diodes (LEDs) as an energy source in Chlorella fusca and Synechococcus nidulans cultures. Bioresour. Technol., 247:1242-1245.

Eiser A.R. 2017. Why does Finland have the highest dementia mortality rate? Environmental factors may be generalizable. Brain Res. 1671:14-17.

United States. 1996. Safe Drinking Water Act Amendments of 1996. Pub.L. 104–182, 110 Stat. 1613.

EPA. 2016. Water Treatment Optimization for Cyanotoxins. EPA 810-B-16-007.

140

EPA. 2019. Cyanobacteria and Cyanotoxins: Information for Drinking Water Systems. EPA-810F11001.

Ersmark K., Del Valle J.R., and Hanessian S. 2008. Chemistry and Biology of the Aeruginosin Family of Serine Protease Inhibitors. Angew. Chem Int. Ed. 47:1202-1223.

Falconer I.R., and Humpage A.R. Tumour promotion by cyanobacterial toxins. Phycologia. 1996. 35(6 suppl): 74-79.

Falconer I.R. 1998. Algal toxins and human health. In: Hrubec J (ed.) Quality and treatment of drinking water n. The handbook of environmental chemistry Vol. 5, Part C:53-82.

Fawell J.K., Mitchell R.E., Everett D.J., and Hill R.E. The toxicity of cyanobacterial toxins in the mouse: I microcystin-LR. Hum. Exp. Toxicol. 1999 Mar: 18(3):162-7.

Feurstein D., Holst K., Fischer A., and Dietrich D.R. 2009. Oatp-associated uptake and toxicity of microcystins in primary murine whole brain cells. Toxicol Appl Pharmacol. 234(2):247–255.

Fewer D. P., Köykkä M., Halinen K., Jokela J., Lyra, C. and Sivonen K. 2009. Culture-independent evidence for the persistent presence and genetic diversity of microsystin-producing Anabaena (Cyanobacteria) in the Gulf of Finland. Environ. Microbiol. 11:855–66.

Fischer W.J., Altheimer S., Cattori V., Meier P.J., Dietrich D.R., and Hagenbuch B. 2005. Organic anion transporting polypeptides expressed in liver and brain mediate uptake of microcystin. Toxicol. Appl. Pharmacol., 203 (3):257-263.

Fleming L.E., Rivero C., Burns J., Williams C., Bean J.A., Shea K.A., and Stinn J. 2002. Blue green algal (cyanobacterial) toxins, surface drinking water, and liver cancer in Florida. Harmful Algae. 1(2):157-168.

Francy D.S., Brady A.M.G., Ecker C.D., Graham J.L., Stelzer E.A., Struffolino P., Dwyer D.F., and Loftin K.A. 2016. Estimating microcystin levels at recreational sites in western Lake Erie and Ohio. Harmful Algae. 58:23-34.

Gehringer M.M. 2004. Microcystin-LR and okadaic acid-induced cellular effects: a dualistic response. FEBS Letters. 557(1-3):1-8.

Gobler C.J., Davis T.W., Coyne K.J., and Boyer G.L. 2007. Interactive influences of nutrient loading, zooplankton grazing and microcystin synthetase gene expression on cyanobacterial bloom dynamics in a eutrophic New York lake. Harmful Algae. 6:119–133.

Graham D., Kisch H., Lawton L.A., and Robertson P.K.J. 2010. The degradation of microcystin-LR using doped visible light absorbing photocatalysts. Chemosphere. 78:1182-5.

Griffith A.W., Doherty O.M., and Gobler C.J. 2019. Ocean warming along temperate western boundaries of the Northern Hemisphere promotes an expansion of Cochlodinium polykrikoides blooms. Proc. R. Soc. B 286.

141

Guedes I.A., Rachid C.T.C.C., Rangel L.M., Silva L.H.S., Bisch P.M., Azevedo S.M.F.O. and Pacheco A.B.F. 2018. Close Link Between Harmful Cyanobacterial Dominance and Associated Bacterioplankton in a Tropical Eutrophic Reservoir. Front. Microbiol. 9:424.

Harris T.D., and Graham J.L. 2017. Predicting cyanobacterial abundance, microcystin, and geosmin in a eutrophic drinking-water reservoir using a 14-year dataset. Lake and Reservoir Management. 33(1):32-48.

Hawkins P.R., Runnegar M.T.C., Jackson A.R.B., and Falconer I.R. 1985. Severe hepatotoxicity caused by the tropical cyanobacterium (blue-green alga) Cylindrospermopsis raciborskii (Wolozynska) Seenaya and Subba Raju isolated from a domestic water supply reservoir. Appl. Env. Microbiol., 50:1292–1295.

Heck K., Alvarenga D.O., Shishido T.K., Varani A.M., Dorr F.A., Pinto E., Rouhiainen L., Jokela J., Sivonen K., and Fiore M.F. 2018. Biosynthesis of microcystin heptatotoxins in the cyanobacterial genus Fischerella. Toxicon. 141:43-50.

Heisler J., Glibert P.M., Burkholder J.M., Anderson D.M., Cochlan W., Dennison W.C., Dortch Q., Gobler C.J., Heil C.A., Humphries E., Lewitus A., Magnien R., Marshall H.G., Sellner K., Stockwell D.A., Stoecker D.K., and Suddleson M. 2008. Eutrophication and harmful algal blooms: a scientific consensus. Harmful Algae, 8(1):3-13.

Holdren G.C., and Turner K. 2010. Characteristics of Lake Mead, Arizona–Nevada: Lake and Reservoir Management. 26(4):230-239.

Holland P.M., Abramson R.D., Watson R., and Gelfand, D.H. 1991. Detection of specific polymerase chain reaction product by utilizing the 5'----3' exonuclease activity of Thermus aquaticus DNA polymerase. Proceedings of the National Academy of Sciences of the United States of America. 88(16):7276–7280.

Holmes C., and Cotterell D. 2009. Role of infection in the pathogenesis of Alzheimer’s disease: implications for treatment. CNS Drugs. 23:993–1002.

Holtcamp, W. 2012. The Emerging Science of BMAA: Do Cyanobacteria Contribute to Neurodegenerative Disease? Environmental Health Perspectives. 120(3).

Huisman J, Matthijs H.C.P., and Visser P.M. 2005. Harmful Cyanobacteria. Springer, Berlin.

Humpage A.R., Hardy S.J., Moore E.J., Froscio S.M., and Falconer I.R. 2000. Microcystins (cyanobacterial toxins) in drinking water enhance the growth of aberrant crypt foci in the mouse colon. Journal of Toxicology and Environmental Health, Part A. 61:155-165.

Inoue-Sakamoto, K., Tanji, Y., Yamaba, M., Natsume T., Masaura T., Asano T., Nishiuchi T., and Sakamoto T. 2018. Characterization of extracellular matrix components from the desiccationtolerant cyanobacterium Nostoc commune. J. Gen. Appl. Microbiol. 64:15–25.

Jackim E., and Gentile J.H. 1968. Toxins of blue-green algae: Similar to saxitoxin. Science. 162:916-916.

142

Jacoby J.M., Collier D.C., Welch E.B., Hardy F.J., and Crayton M. 2000. Environmental factors associated with a toxic bloom of Microcystis aeruginosa. Canadian Journal of Fisheries and Aquatic Sciences 57:231–240.

Jacoby J., Burghdoff M., Williams G., Read L., and Hardy J. 2015. Dominant factors associated with microcystins in nine midlatitude, maritime lakes. Inland Waters. 5(2):187-202.

Jonasson S., Eriksson J., Berntzon L., Spácil Z., Ilag L.L., Ronnevi L.O., Rasmussen U., and Bergman B. 2010. Transfer of a cyanobacterial neurotoxin within a temperate aquatic ecosystem suggests pathways for human exposure. Proc. Natl. Acad. Sci. U.S.A, 107:9252-9257.

Jungblut A.D., and Neilan B.A. 2006. Molecular identification and evolution of the cyclic peptide hepatotoxins, microcystin and nodularin, synthetase genes in three orders of cyanobacteria. Arch Microbiol. 185:107-114.

Kaebernick M., Neilan B.A., Borner T., and Dittmann E. 2000. Light and the transpirational response of the microcystin biosynthesis gene cluster. Appl. Environ. Microbiol. 66:3387–3392.

Kaebernick M., Dittmann E., Borner T., Neilan B.A. 2002. Multiple alternate transcripts direct the biosynthesis of microcystin, a cyanobacterial nonribosomal peptide. Appl Environ Microbiol. 68:449-455.

Kotak B.G., Lam A.K.Y., Prepas E.E., and Hrudey S.E. 2000. Role of chemical and physical variables in regulating microcystin-LR concentration in phytoplankton of eutrophic lakes. Canadian Journal of Fisheries and Aquatic Sciences. 57:1584-1593.

Kuiper-Goodman T., Falconer I., and Fitzgerald J. 1999. Human health aspects. In: Chorus I, Bartram J, editors. Toxic Cyanobacteria in Water: A Guide to Their Public Health Consequences, Monitoring, and Management. E & FN Spoon; London:113–153.

Kurland L.T. and Mulder D.W. Epidemiologic Investigations of Amyotrophic Lateral Sclerosis. (1954) Neurology. 4:355-378.

Kurmayer R., Christiansen G., and Chorus I. 2003. The abundance of microcystin-producing genotypes correlates positively with colony size in Microcystis and determines its microcystin net production in Lake Wannsee. Applied and Environmental Microbiology. 69:787–795.

LaBounty J.F., and Horn M.J. 1997. The influence of drainage from the Las Vegas Valley on the limnology of Boulder Basin, Lake Mead, Arizona-Nevada. Lake Reserv. Manage. 13:95-108.

LaBounty J.F., and Burns N.M. 2005. Characterization of boulder basin, Lake Mead, Nevada-Arizona, USA – Based on analysis of 34 limnological parameters. Lake Reserv. Manage. 21:277-307.

LaBounty J.F., and Burns N.M. 2007. Long-term increases in oxygen depletion in the bottom water of Boulder Basin, Lake Mead, Nevada-Arizona, USA. Lake Reserv Manage. 23:69-82.

143

Lee T.A., Rollwagen-Bollens G., Bollens S.M., and Faber-Hammond J.J. 2015. Environmental influence on cyanobacteria abundance and microcystin toxin production in a shallow temperate lake. Ecotoxicology and Environmental Safety. 114:318-325.

Li H., Xu J., Liu Y., Ai S., Qin F., Li Z., Zhang H., and Huang Z. 2011. Antioxidant and moisture-retention activities of the polysaccharide from Nostoc commune. Carbohydrate Polymers. 83:1821–1827.

Li X.B., Zhang X., Ju J., Li Y., Yin L., and Pu Y. 2014. Alterations in neurobehaviors and inflammation in hippocampus of rats induced by oral administration of microcystin-LR. Environ Sci Pollut Res. 21:12419–12425.

Lun Z., Hai Y., and Kun C. (2002). Relationship between microcystin in drinking water and colorectal cancer. Biomed. Environ. Sci. 15:166–171.

MacKintosh C., Beattie K.A., Klumpp S., Cohen P., and Codd G.A. 1990. Cyanobacterial microcystin-LR is a potent and specific inhibitor of protein phosphatases 1 and 2A from both mammals and higher plants. FEBS Letters. 264:187–192.

Mazur H., and Pliński M. 2003. Nodularia spumigena blooms and the occurrence of hepatotoxin in the Gulf of Gdańsk. Oceanologia. 45:305–316.

Meissner K.E., Dittmann E., and Borner T. 1996. Toxic and non-toxic strains of the cyanobacterium Microcystis aeruginosa contain sequences homologous to peptide synthetase genes. FEMS Microbiology Letters 135:295-303.

Mikalsen B., Boison G., Skulberg O.M.G., Fastner J., Davies W., Gabrielsen T.M., Rudi K., and Jakobsen K.S. 2003. Natural variation in the microcystin synthetase operon mcyABC and impact on microcystin production in Microcystis strains. J Bacteriol. 185(9):2774-85.

Monchamp M.E., Pick F.R., Beisner B.E., and Maranger R. 2014. Nitrogen forms influence microcystin concentration and composition via changes in cyanobacterial community structure. PLoS ONE, 9(1).

Mondo K., Hammerschlag N., Basile M., Pablo J., Banack S.A., and Mash D.C. 2012. Cyanobacterial neurotoxin β-N-methylamino-L-alanine (BMAA) in shark fins. Marine Drugs. 10:509-520.

Mosley L.M. 2015. Drought impacts on the water quality of freshwater systems; review and integration. Earth Sci. Rev. 140:203-214.

Mowe M.A.D., Mitrovic S.M., Lim R.P., Furey A., and Yeo D.C.J. 2015. Tropical cyanobacterial blooms: a review of prevalence, problem taxa, toxins and influencing environmental factors. J. Limnol. 74:205–224.

Murch S., Cox P., Banack S. 2004. A mechanism for slow release of biomagnified cyanobacterial neurotoxins and neurodegenerative disease in Guam. PNAS. 101(33):12228-12231.

144

Nabout J.C., da Silva Rocha B., Carneiro F.M., Sant’Anna C.L. 2013. How many species of Cyanobacteria are there? Using a discovery curve to predict the species number. Biodivers Conserv. 22:2907–2918.

Nadkarni M. A., F. E. Martin, N. A. Jacques, and N. Hunter. 2002. Determination of bacterial load by real-time PCR using a broad-range (universal) probe and primers set. Microbiology. 148:257-266.

Neilan B.A., Jacobs D., Blackall L.L., Hawkins P.R., Cox P.T., Goodman A.E. 1997. rRNA sequences and evolutionary relationships among toxic and nontoxic cyanobacteria of the genus Microcystis. Int J Syst Bacteriol. 47(3):693–697.

Nishiwaki-Matsushima R., Ohta T., Nishiwaki S., Suganuma M., Kohyama K., Ishikawa T., Carmichael W.W., Fujiki H. 1992. Liver tumor promotion by the cyanobacterial cyclic peptide toxin microcystin-LR. J. Cancer Res. Clin. Oncol. 118:420-424.

Nonneman D., Zimba P. 2002. A PCR-Based Test to Assess the Potential for Microcystin Occurrence in Channel Catfish Production Ponds. Journal of Phycology. 38(1):230-233.

Nubel U., Garcia-Pichel F., Muyzer G. 1997. PCR primers to amplify 16S rRNA genes from cyanobacteria. Appl Environ Microbiol. 63: 3327–3332.

Olson, J. M. 2006. Photosynthesis in the Archean era. Photosynth. Res. 88:109–117.

Orihel, D.M., Bird, D.F., Brylinsky, M., Chen, H., Donald, D.B., Huang, D.Y., Giani, A., Kinniburgh, D., Kling, H., Kotak, B.G., Leavitt, P.R., Nielsen, C.C., Reedyk, S., Rooney, R.C., Watson, S.B., Zurawell, R.W., and Vinebrooke, R.D. 2012. High microcystin concentrations occur only at low nitrogen-tophosphorus ratios in nutrient-rich Canadian lakes. Can. J. Fish. Aquat. Sci. 69(9):1457–1462.

Pablo J., Banack S.A., Cox P.A., Johnson T.E., Papapetropoulos S., Bradley W.G., Buck A., Mash D.C. 2009. Cyanobacterial neurotoxin BMAA in ALS and Alzheimer’s disease. Acta Neurol Scand. 120(4):216-25.

Paerl H. 2008. Nutrient and other environmental controls of harmful cyanobacterial blooms along the freshwater-marine continuum. Advances in Experimental Medicine and Biology. 619:216-241.

Paerl H., Huisman J. 2009. Climate change: a catalyst for global expansion of harmful cyanobacteria blooms. Environ Microbiol Rep. 1(1):27-37.

Paerl H., Paul V. 2012. Climate change: links to global expansion of harmful cyanobacteria. Water Res. 46(5):1349-63.

Paerl H., Otten T. 2013. Harmful cyanobacterial blooms: causes, consequences, and controls. Microb Ecol. 65(4):995-1010.

Pearson L., Dittman E., Mazmouz R., Ongley S., D’Agostino P., Neilan B. 2016. The genetics, biosynthesis and regulation of toxic specialized metabolites of cyanobacteria. Harmful Algae. 54:98-111.

145

Pan H., Song L., Liu Y., Borner T. 2002. Detection of hepatatoxic Microcystis strains by PCR with intact cells from both culture and environmental samples. Arch. Microbiol. 178(6):421-7.

Pick F.R. 2016. Blooming algae: a Canadian perspective on the rise of toxic cyanobacteria. Can. J. Fish. Aquat. Sci., 73:1149-1158.

Proctor E.A., Mowrey D.D., and Dokholyan N.V. 2019. β-Methylamino-L-alanine substitution of serine in SOD1 suggests a direct role in ALS etiology. PLoS Comput Biol 15(7).

Purvina, S., Bechemin, C., Balode, M., Grzebyk, D. and Maestrini, S. 2008. The influence of inorganic nutrients and dissolved organic matter on the growth of cyanobacteria Microcystis aeruginosa isolated from the Gulf of Riga. Acta Univ. Latv. Biol. 745:61-74.

Radau, G., Schermuly, S. and Fritsche, A. 2003. New cyanopeptidederived low molecular weight inhibitors of trypsin-like serine proteases. Archiv der Pharmazie, 336:300–309.

Rastogi R.P., Madamwar D. and Incharoensakdi A. 2015 Bloom Dynamics of Cyanobacteria and Their Toxins: Environmental Health Impacts and Mitigation Strategies. Front. Microbiol. 6:1254.

Recknagel, F. 1997. ANNA–Artificial Neural Network model for predicting species abundance and succession of blue-green algae. Hydrobiologia 349:49–59.

Rinta-Kanto, J. M., Ouellette A.J.A., Boyer G.L., Twiss M.R., Bridgeman T.B., and Wilhelm S.W. 2005. Quantification of toxic Microcystis spp. during the 2003 and 2004 blooms in western Lake Erie using quantitative real-time PCR. Environmental Science & Technology 39:4198-4205.

Rinta-Kanto J.M., Konopko E.A., DeBruyn J.M., Bourbonniere R.A., Boyer G.L., Wilhelm S.W. 2009. Microcystis: relationship between microcystin production, dynamics of genotypes and environmental parameters in a large lake. Harmful Algae. 8:665-673.

Rodgers K.J., Dunlop R. 2011. The cyanobacteria-derived neurotoxin BMAA can be incorporated into cell proteins and could thus be an environmental trigger for ALS and other neurological diseases associated with protein misfolding. Presented at: 22nd Annual Symposium on ALS/MND, Sydney, Australia.

Rosen M.R., Turner K., Goodbred S.L., and Miller J.M. 2012, A synthesis of aquatic science for management of Lakes Mead and Mohave: U.S. Geological Survey Circular 1381, 162 p.

Salomonsson M.L., Fredriksson E., Alfjorden A., Hedeland M., and Bondesson U. 2015. Seafood sold in Sweden contains BMAA: A study of free and total concentrations with UHPLC–MS/MS and dansyl chloride derivatization. Toxicology Reports 2:1473-1481.

Schopf J.W. 1993. Microfossils of the Early Archean Apex chert: New evidence of the antiquity of life. Science. 260:640–646.

146

Scholz S.N., Esterhuizen-Londt M., and Pflugmacher S. 2017. Rise of toxic cyanobacterial blooms in temperate freshwater lakes: causes, correlations and possible countermeasures. Toxicol Environ Chem. 99:543–577.

Schulze P.S.C, Barreira L.A., Pereira H.G.C., Perales J.A., and Varela J.C.S. 2014. Light emitting diodes (LEDs) applied to microalgal production. Trends Biotechnol., 32:422-430.

Scott L.L., and Downing T.G. 2018. A single neonatal exposure to BMAA in a rat model produces neuropathology consistent with neurodegenerative diseases. Toxins. 10(1):22.

Scott J.T., McCarthy M.J., Otten T.G., Steffen M.M., Baker B.C., Grantz E.M., Wilhelm S.W., Paerl H.W. 2013. Comment: An alternative interpretation of the relationship between TN:TP and microcystins in Canadian lakes. Can. J. Fish. Aquat. Sci. 70:1265–1268.

Scott L.L., Downing S., Phelan R.R., and Downing T.G. 2014. Environmental modulation of microcystin and β-N-methylamino-L-alanine as a function of nitrogen availability. Toxicon. 87:1-5.

Scott L.L., Downing S., and Downing T. 2018. Potential for dietary exposure to β-N-methylamino-L-alanine and microcystin from a freshwater system. Toxicon. 150:261-266.

Semenova A.S., Sidelev S.I., and Dmitrieva O.A. 2017. Experimental Investigation of Natural Populations of Daphnia galeata G.O. Sars from the Curonian Lagoon Feeding on Potentially Toxigenic Cyanobacteria. Biology Bulletin. 44(5):538-546.

Sevilla E., Martin-Luna B., Vela L., Bes M.T., Fillat M.F., and Peleato M.L. 2008. Iron availability affects mcyD expression and microcystin-LR synthesis in Microcystis aeruginosa PCC7806. Environ. Microbiol. 10:2476-2483.

Sivonen K., Leikoski N., Fewer D.P., and Jokela J. 2010. Cyanobactins-ribosomal cyclic peptides produced by cyanobacteria. Appl Microbiol Biotechnol. 86:1213-1225.

Sirisuk P., Sunwoo I., Kim S.H., Awah C.C., Ra C.H., Kim J.M., Jeong G.T., and Kim S.K. 2018. Enhancement of biomass, lipids, and polyunsaturated fatty acid (PUFA) production in Nannochloropsis oceanica with a combination of single wavelength light emitting diodes (LEDs) and low temperature in a three-phase culture system. Bioresource Technology. 270:504-511.

Sipari H., Rantala-Ylinen A., Jokela J., Oksanen I., and Sivonen K. 2010. Development of a chip assay and quantitative PCR for detecting microcystin synthetase E gene expression. Appl. Environ. Microbiol. 76:3797–3805.

Sivonen K., and Jones G. 1999. Cyanobacterial Toxins. Toxic Cyanobacteria in Water. E&FN Spon, London, pp. 41-111.

Snyder S.A., Keith T.L., Verbrugge D.A., Snyder E.M., Gross T.S., Kannan K., and Giesy J.P. 1999. Analytical methods for detection of selected estrogenic compounds in aqueous mixtures: Environmental Science and Technology. 33(16):2814–2820.

147

Spencer P., Nunn P., Hugon J., Ludolph A., Ross S., Roy D., and Robertson R. 1987. Guam amylotrophic lateral sclerosis-parkinsonism-dementia linked to a plant excitant neurotoxin. Science. 237(4814):517-22.

Spoof L., and Catherine A. 2017. Appendix 3. Tables of microcystins and nodularins. In: Meriluoto J, Spoof L, Codd GA (eds) Handbook of cyanobacterial monitoring and cyanotoxin analysis. John Wiley & Sons, Chichester, pp 526–537.

Stewart I., Seawright A.A., and Shaw G.R. 2008. Cyanobacterial poisoning in livestock, wild mammals and birds – an overview. In: Hudnell H.K. (eds) Cyanobacterial Harmful Algal Blooms: State of the Science and Research Needs. Advances in Experimental Medicine and Biology, vol 619.

Testai E., Buratti F.M., Funari E., Manganelli M., and Vichi S. 2016. Review and analysis of occurrence, exposure and toxicity of cyanobacteria toxins in food. EFSA supporting publication 2016:EN-998. 309 pp.

Tietjen T. 2015. Microcystis in Lakes Mead and Mohave: Winter 2014-2015. Southern Nevada Water Authority.

Tilahun S., Kifle D., Zewde T.W., Johansen J.A., Demissie T.B., and Hansen J.H. 2019. Temporal dynamics of intra-and extra-cellular microcystins concentrations in Koka Reservoir (Ethiopia): implications for public health risk. Toxicon. 168:83–93.

Tomitani A., Knoll A.H., Cavanaugh C.M., and Ohno T. 2006. The evolutionary diversification of cyanobacteria: Molecular phylogenetic and palaeontological perspectives. Proc Natl Acad Sci USA. 103:5442-5447.

Tonk L., Visser P.M., Christiansen G., Dittmann E., Snelder E.O.F.M., Wiedner C., Mur L.R., and Huisman J. 2005. The Microcystin Composition of the Cyanobacterium Planktothrix agardhii changes toward a More Toxic Variant with Increasing Light Intensity. Applied and Environmental Microbiology. 71(9):5177-5181.

Urbach E., Robertson D.L., and Chisholm S.W. 1992. Multiple evolutionary origins of prochlorophytes within the cyanobacterial radiation. Nature. 355:267-270.

Uyeda J.C., Harmon L.J., and Blank C.E. 2016. A Comprehensive Study of Cyanobacterial Morphological and Ecological Evolutionary Dynamics through Deep Geologic Time. PLoS ONE 11(9).

Vaitomaa J., Rantala A., Halinen K., Rouhiainen L., Tallberg P., Mokelke L., and Sivonen K. 2003. Quantitative real-time PCR for determination of microcystin synthetase E copy numbers for Microcystis and Anabaena in lakes. Applied and Environmental Microbiology. 69:7289-7297.

Vijayakumar S., and Menakha M. 2015. Pharmaceutical applications of cyanobacteria – A review. Journal of Acute Medicine. 5(1):15-23.

148

Wawrik B., Callaghan A. V., and Bronk D. A. 2009. Use of inorganic and organic nitrogen by Synechococcus spp. and Diatoms on the West Florida Shelf as measured using stable isotope probing. Appl Environ Microbiol, 75:6662-6670.

Welker M., Brunke M., Preussel K., Lippert I., and von Döhren H. 2004. Diversity and distribution of Microcystis (Cyanobacteria) oligopeptide chemotypes from natural communities studied by single-colony mass spectrometry. Microbiology-Sgm, 150:1785-1796.

Welker M., and von Döhren H. 2006. Cyanobacterial peptides - nature's own combinatorial biosynthesis FEMS Microbiol. Rev. 30(4):530-563.

Wert E.C., Dong M.M., and Rosario-Ortiz F.L. 2013. Using digital flow cytometry to assess the degradation of three cyanobacteria species after oxidation processes Water Res. 47(11):3752-3761. Whiting M. 1988. Toxicity of cycads: implications for neurodegenerative diseases and cancer. Third World Medical Research Foundation.

Whitton B.A. 2012. Ecology of Cyanobacteria II. Their diversity in Space and Time.

Whitton B.A, and Potts M. 2002. The Ecology of Cyanobacteria. Their Diversity in Time and Space.

World Health Organization. 2003. Cyanobacterial toxins: Microcystin-LR in Drinking-water. 2nd ed. Addendum to Vol. 2 Health criteria and other supporting information.

Wood S.A., Dietrich D.R., Cary S.C., and Hamilton D.P. 2012. Increasing Microcystis cell density enhances microcystin synthesis: a mesocosm study. Inland Waters. 2:17-22.

Wynne T.T., and Stumpf R.P. 2015. Spatial and temporal patterns in the seasonal distribution of toxic cyanobacteria in Western Lake Erie from 2002-2014. Toxins, 7(5):1649-1663.

Zamyadi, A. MacLeod S.L., Fan Y., McQuaid N., Dorner S., Sauve S., and Prevost M. 2012. Toxic cyanobacterial breakthrough and accumulation in a drinking water plant: A monitoring and treatment challenge. Water Research. 46:1511-1523.

Zastepa, A., Pick, F.R., and Blais, J.M. 2014. Fate and persistence of particulate and dissolved microcystin-LA from Microcystis blooms. Hum. Ecol. Risk Assess. 20:1670–1686.

Zegura B., Straser A., and Filipic M. 2011. Genotoxicity and potential carcinogenicity of cyanobacterial toxins – a review. Mutation Research. 727:16-41.

Zhou L., Yu H., and Chen K. 2002. Relationship between microcystin in drinking water and colorectal cancer. Biomed. Environ. Sci. 15:166-171.

Zhang M., Duan H., Shi X., Yu Y., and Kong F. 2012. Contributions of meteorology to the phenology of cyanobacterial blooms: Implications for future climate change Water Res. 46:442-452.

149

Zimmerman H.M. 1945. Monthly report to the medical officer in command. United States Navy Medical Research Unit No. 2. June, Dayton, OH, USA.

150

CURRICULUM VITAE

TIMOTHY W. ALBA

Graduate Student School of Life Sciences

University of Nevada Las Vegas Las Vegas, NV 89119

702-401-0766 [email protected]

EDUCATION

University of Nevada Las Vegas 2012-2020 PhD Student (Discipline: Microbiology) Las Vegas, NV Development of an innovative approach to detecting toxin-producing cyanobacteria

University of Washington 2008-2010 MS in Civil Engineering Seattle, WA Enhanced biogas production through anaerobic co-digestion of organic wastes

Touro University International 2006-2009 MBA, Public Management Cypress, CA

University of Washington 2001-2003 BS in Civil Engineering Seattle, WA Biological removal of phosphorus and nitrogen from wastewater

Skagit Valley College 1998-2000 Pre-engineering Mount Vernon, WA

Sehome High School 1994-1998 Diploma Bellingham, WA

EMPLOYMENT HISTORY

CDM 2010-2011 Environmental Engineer Phoenix, AZ

Assisted with civil and process mechanical design of wastewater treatment plant upgrades

Provided construction services and quality assurance inspections at a treatment plant Drafted a report on anaerobic co-digestion of waste products like fats, oils, and greases

HDR 2009 Water/Wastewater Intern Bellevue, WA

Conducted a treatability study of Alaska well water for color and odor removal

151

Drafted a report on horizontal directional drilling feasibility for a combined sewer overflow study

MWH/University of Washington 2003 Research Assistant Boise, ID

Constructed, operated, and sampled a step-feed biological nutrient removal pilot plant

US Army 2003-2008 Corps of Engineers Officer Germany/Iraq

Supervised combat operations in Iraq Supervised pre-deployment training in Germany

US Army Corps of Engineers 2002 Cost estimating intern New Orleans, LA

Performed cost estimates on dredging and levee improvement projects

PEER-REVIEWED PUBLICATIONS

Hedlund, B.P., Murugapiran, S.K., Alba, T.W., Levy, A., Dodsworth, J.A., Goertz, G.B., Ivanova, N., Woyke, T. 2015. Uncultivated thermophiles: Current status and spotlight on ‘Aigarchaeota’. Current Opinion in Microbiology. 25: 136-145.

HONORS, FELLOWSHIPS, & AWARDS

UNLV Graduate College Hermsen Fellowship. 2014. Environmental Engineer of the Future Scholarship. 2008. US Army Bronze Star Medal. 2006. Sapper Leader Course Graduate. 2004. US Army ROTC Distinguished Military Graduate. 2003. UW Civil Engineering Charles Church More Scholarship. 2001. National Guard Association of Washington Scholarship. 2001. US Army Signal Support Systems Specialist AIT Honor Graduate. 2001. Irene Schumaker Scholarship. 1999.

MEETING PRESENTATIONS

Alba, T.W., Murugapiran, S.K., Levy, A., Dodsworth, J.A., Goertz, G.B., Ivanova, N., Woyke, T., Hedlund, B.P. 2015. Shedding light on ‘Aigarchaeota’, an uncultivated lineage of obligate thermophiles. JGI User Meeting. March 24-26, 2015. Walnut Creek, CA. Poster presentation. Alba, T.W., Murugapiran, S.K., Blainey, P.C., Dodsworth, J.A., Thomas, S., Woyke, T., Rinke, C., DeVlaminck, I., Stepanauskas, R., Quake, S.R., Hedlund, B.P. 2014. Insigths into the Global Diversity and Physiology of ‘Aigarchaeota’ through Synergistic Analysis of Single-Cell Genomes and Metagenomes. JGI User Meeting. March 18-20, 2014. Walnut Creek, CA. Poster presentation.

152

Alba, T.W. 2014. Insights into the Global Diversity and Physiology of ‘Aigarchaeota’ through Synergistic Analysis of Single-Cell Genomes and Metagenomes. ASU Hot Life in the Desert Meeting #9. March 5-7, 2014. Tempe, AZ. Oral presentation. Alba, T.W., Goertz, G.B., Williams, A.J., Cole, J.K., Murugapiran, S.K., Dodsworth, J.A., Hedlund, B.P.2013. Diversity and Habitat Niche Modeling of Candidate Archaeal Phylum ‘Aigarchaeota’. American Geophysical Union Fall Meeting. December 9-13, 2013. San Francisco, CA. Poster presentation.

TEACHING EXPERIENCE

Substitute Lecturer 2014, 2015, 2017 UNLV School of Life Sciences Las Vegas, NV Course taught: BIOL 251 (Microbiology) 100+ student lecture

Teaching Assistant 2012-2020 UNLV School of Life Sciences Las Vegas, NV Courses taught: BIOL 189 lab, BIOL 251 lab, BIOL 351 lab 20+ student lecture

PROFESSIONAL CERTIFICATION

Engineer-In-Training, State of Washington. 2003.

Documents

Rapid Detection of Toxin-Producing Cyanobacteria