Analysis of Life Cycle within Various Strains of Cyanobacteria with a Focus on Internal Regulators & Toxin Production Jackie Fischer1,2 , Dr. Heath Mash3, Christina Bennett-Stamper3, and Dr. Dion Dionysiou1 1. University of Cincinnati, Dept. of Environmental and Chemical Engineering, 2. Pegasus Technical Services Inc., 3. U.S. Environmental Protection Agency

Can internal regulators be used to Can internal regulator measurements be used to What external factors contribute Conclusions Introduction indicate/predict life cycle phase shifts? predict toxin production and/or cellular release to these stage shifts? Using anabaena circinalis: Phase I Cyanobacteria are photosynthetic bacteria that of the toxins? Are they required? Thresholds? Nitrate concentrations exhibit some similarities to algae and can be How do these parameters contribute to and work in partnership with or against each other to progress found naturally in lakes, streams, ponds, and other . Nitrogen spiked- gradual decrease until cell cyanobacterial cells through their life cycle shifts and into producing and releasing toxins? surface waters1. However, toxin producing morphology shifted from vegetative cells to cyanobacteria have become an increasing concern Phase I • Results Phase I • Imaging Phase II vegetative cells with heterocysts as growth rates have been escalating. Nevertheless, the main triggering factors Phosphate Spiked Samples Cell Density . Phosphorous spiked- inconsistent and controlling these increased growth rates are not 7.00E+07 fluctuating concentration with heterocysts 6.00E+07 fully understood1,2. As such, it is of paramount forming within the first week of growth. 5.00E+07

concern to learn more about these bacteria to 4.00E+07 Phosphate concentrations

allow for accurate prediction of toxin synthesis cells/L 3.00E+07 B1 . Nitrogen spiked- oscillating concentration B3 and potential neutralization solutions to avoid 2.00E+07 B4 mass toxin release into waterways. 1.00E+07 Figure 5: Growth Experiments for isolating and manipulating . Phosphorous spiked- gradually decreasing 0.00E+00 cell types over time 11/2/2016 11/4/2016 11/6/2016 11/8/2016 12/2/2016 12/4/2016 12/6/2016 12/8/2016 11/10/2016 11/12/2016 11/14/2016 11/16/2016 11/18/2016 11/20/2016 11/22/2016 11/24/2016 11/26/2016 11/28/2016 11/30/2016 Dates Cell Densities Procedure Figure 1: Phosphate Spiked samples with a. circinalis Image !: Aggregates of a. circinalis displaying forming heterocysts . Phosphorous spiked sample- lower overall Strains: anabaena circinalis, anabaena flos aquae, and (Figure 1) as compared to their nitrogen spiked Nitrate Spiked Samples Cell Density microcysts aeruginosa 7.00E+07 counterparts (Figure 2) Phase I: Initial experiments 6.00E+07  Cell strands were much longer, with several 5.00E+07 heterocysts per filament. Examine the influence of nutrient availability on 4.00E+07

life cycle progression via alteration of cells/L 3.00E+07 A1 A2 . Both nutrient spiked samples- consistently nitrogen/phosphorus concentrations to assess 2.00E+07 A3 denser than the two controls (Figure 3). their impact on toxin production, cell integrity, 1.00E+07 A4 0.00E+00 and cellular growth dynamics. Image 3: Growth Experiments for isolating and manipulating vegetative cells Next Steps 11/2/2016 11/4/2016 11/6/2016 11/8/2016 12/2/2016 12/4/2016 12/6/2016 12/8/2016 11/10/2016 11/12/2016 11/14/2016 11/16/2016 11/18/2016 11/20/2016 11/22/2016 11/24/2016 11/26/2016 11/28/2016 11/30/2016 Determine cell density and morphology to Dates Phase I: Growth Curves (In Triplicate) Internal energy Figure 2: Nitrate Spiked samples with a. circinalis provide growth curves for each nutrient spiking Figure 4: Cyanobacteria Life Cycle model (CMC) by Analysis Notes Frequency Hense and Beckmann (2006)3. Nutrients Phosphates, Nitrates Daily . Luminometer & ATP assay kit scenario. Cell Densities for the Controls: Conditions pH, Temperature, Light intensity Daily Nutrient Starvation & Abundance Scenarios Cell Counts Cell Density Weekly  Free internal energy before & after 4.00E+07 Cell Morphology Weekly Phase II: (Life Cycle Isolation/Manipulation) extended periods of dark at each life cycle Phase II: Cell Isolation 3.50E+07 Analysis Notes Frequency 3.00E+07 phase. Take base parameters from culture flasks and Nutrients Phosphates, Nitrates Daily 2.50E+07 Conditions pH, Temperature, Light intensity Daily L/llse Cell Counts Cell Density Weekly compare to nutrient manipulated subcultures. 2.00E+07 Intercellular nitrogen

c Cell Morphology Weekly 1.50E+07 Starvation ELISA AND LC/MS Extracellular and Total Toxin Concentrations Each Life Cycle Phase . Modified American Society of Testing This allows a step-wise analysis of the internal 1.00E+07 Abundance Transmission Electron Examine Cell Membrane Each Life Cycle Materials (ASTM) method (E1757-01B 5.00E+06 Microscope Phase regulators and nutrient constraints necessary for Scanning Electron Microscope Observe the Topography of Cell Surfaces Each Life Cycle 3 0.00E+00 (2015) International preparation of biomass 16 16 16 16 16 16 16 16 Phase 016 016 016 016 016 016 016 progressing a cell through its life cycle. 016 016 016 016 20 20 20 2 2 2 2 2 2 2 2 2 2 2 Spectrophotometer/Colorimeter Chlorophyll and Phycocyanin Each Life Cycle

2/20 4/ 6/ 8/ for compositional analysis) / / / / /2/20 /4/20 /6/20 /8/20 18/ 20/ 22/ 24/ 26/ 28/ 30/ /10/ /12/ /14/ /16/ / / / / / / / Measurements Phase 11 11 11 11 12 12 12 12 11 11 11 11 11 11 11 11 11 11 11 Dates Image 2: Single stains of a. circinalis displaying . TOC/TN combustion analyzer Figure 3: Control samples with a. circinalis heterocysts and hormocysts Table 1: Growth Experiments Analysis for Phase I and Phase II

The views expressed in this presentation are those of the authors and do not necessarily reflect the views or policies of the U.S. Environmental Protection Agency. Any data presented are considered provisional. 1. USEPA. (2012). “Cyanobacteria and Cyanotoxins: Information for Drinking Water Systems.” EPA-810F11001. Office of Water. 2. Hense, Inga; Beckmann, Aike. (2006). “Towards a model of Cyanobacteria Life Cycle-Effects of Growing and Resting Stages on Bloom Formation of N2-fixing Species.” ELSEVIER. Ecological Modelling 195 pp. (205-218). 3. ASTM E1757-01(2015), Standard Practice for Preparation of Biomass for Compositional Analysis, ASTM International, West Conshohocken, PA, 2015, www.astm.org