Chemical Profiling of Microalgae Communities Associated with Eelgrass Haley Nickerson, Dr. Cynthia G. Hays, and Dr. James R. Kraly Department of Chemistry and Department of Biology, Keene State College, Keene NH AbstractAnalytical Methods – Liquid Chromatography Extraction Method: Placed Filter paper into extraction tube. Added 3 mL of solvent to the extraction tube. 45% methanol, 45% acetone and 10% DI water. Wrapped in tin foil, placed in freezer overnight. Centrifuged and pipetted into HPLC vials. Three gradient solvent system of: Solvent A : 80:20 methanol : 0.5 M ammonium acetate. Solvent B : 90:10 acetonitrile : water. Solvent C : ethyl acetate. Flow rate of 1 mL/min High pressure is used to pass mobile phase and separate components as move through the column Detection by UV-Vis lamp at 450 nm. Peaks were identified by retention times and standard spikes. Results and Discussion Removed a sonication step to limit degradation of samples. This information can be used to see how the colonization of seagrass differs in different places in Massachusetts. References Pinckney, J.; Micheli, F. Microalgae on seagrass mimics: Does epiphyte community structure differ from live seagrasses. Journal of Experimental Marine Biology and Ecology. 1991, 221, Wright, S.; Jeffrey, S.; Mantoura, R.; Llewellyn, C.; Bjornland, T.; Repeta, D.; Welschmeyer, N. Improved HPLC method for the analysis of chlorophylls and carotenoids from marine phytoplankton. Mar. Ecol. Prog. Ser. 1991, 77, Many different species of microalgae live on the surface of eelgrass leaves, and can limit the growth and survival of the eelgrass. Microalgae are single-celled photosynthetic organisms. Overgrowth by epiphytic microalgae has been implicated in the decline of seagrasses worldwide. Traditionally microalgae have been identified using microscopy, which is both difficult, time consuming and relies on qualitative observations. The purpose of this research project is to develop an analytical method to measure extracted photopigments as a means of microalgae identification. High Performance Liquid Chromatography (HPLC) will be used to separate and measure photopigments chlorophyll a (C), fucoxanthin (F), and zeaxanthin (Z), which are common to many microalgae communities. Calculated ratios of the three photopigments can be used to identify microalgae taxonomy. Results from this project will investigate the relative abundance of different microalgae communities on eelgrass collected from field sites in Massachusetts, and examine how algal community structure varies with eelgrass health. Specific Aims to Project Establish extraction methods for collecting epiphytes from filters. Develop HPLC separation for photopigments. Asses extraction efficiency of standards using HPLC and quantitative calibration. Apply method to field samples for comparative analysis. Acknowledgements Keene State College Chemistry and Biology Departments Dr. James Kraly, Dr. Cynthia Hays, Mr. Brian Moore Limit of Detection LOD = (3  noise)/sensitivity 1 ppb = parts per billion = 1ng/mL Extraction Efficiency Fucoxanthin10 ppb 62% ± 9 Zeaxanthin3 ppb 60% ± 8 Chlorophyll A50 ppb 84% ± 9 An eelgrass community at sea Photo Credit : Mark Rodriguez / Marine Photobank Check reproducibility for epiphytic eelgrass at different depths and locations within the ocean. Keene State College HPLC Calculated RatioBiological Significance [F]/[C]Relative epiphytic diatom biomass. [Z]/[C]Cyanobacteria contribution to total epiphyte biomass. [F]/[Z]Diatom contribution to cyanobacteria epiphytes. Contact: HPLC Separation of Photopigment Standards Replicate analysis of extracted field samples show high reproducibility Comparative analysis of extracted field samples show photopigment variability