ABSTRACT USE OF THE GREEN MICROALGA MONORAPHIDIUM SP. DEK19 TO REMEDIATE WASTEWATER: SALINITY STRESS AND SCALING TO MESOCOSM CULTURES Anthony Robert Kephart, M.S. Department of Biological Sciences Northern Illinois University, 2016 Gabriel Holbrook, Director The purpose of this thesis was to investigate the growth of the green microalga Monoraphidium sp. Dek19 in the context of phycoremediation under conditions representative of wastewater media of a Midwest wastewater treatment facility. Microalgae were grown in effluent collected from four different wastewater streams at the DeKalb Sanitary District (DSD), DeKalb, Illinois, USA in volumes from 1 L to 380 L. It was determined through preliminary modeling of public data that Monoraphidium sp. Dek19 will likely outcompete local heterospecifics in final effluent wastewaters at the DSD 51.8% of the year. It was also determined that neither nitrogen nor phosphorus should become a limiting nutrient throughout the year. Study of the response of Monoraphidium sp. Dek19 to salinity stress was also completed. Salt stress caused a significant increase in lipid accumulation in salt stressed cultures compared to control cultures (37.0-50.5% increase) with a reduction in biomass productivity. Increases in photosynthetic pigments per cell were seen under salt stress, though the ratio of chlorophyll a and b remained constant. Finally, nitrate uptake was not greatly affected by salinity stress except when biomass accumulation became a corollary for uptake (nitrogen saturation). Luxury uptake of phosphate was significantly affected by wastewater salinity (p<0.001). High sodium potentially interrupts phosphate stress response proteins, slowing internalization of phosphate. Phosphate removal was still possible as some protein binding proteins appear to operate independent of sodium. The effect of indole-3-acetic acid on regulation of cell division in Monoraphidium sp. Dek19 was evaluated at a range of concentrations and no significant differences were observed. A comparison of Monoraphidium sp. Dek19 growth in activated sludge, post-primary filtration, and two final wastewater streams revealed a minimum effect of biotic stressors. The microalgae also grew readily in all wastewaters, though initial population density demands are likely different for each effluent type. Monoraphidium sp. Dek19 grown at large scale revealed light availability as more important than other factors even when compared to large temperature changes (10-25 °C). Low-light growth tanks (380 L) showed significantly reduced specific growth rate though microalgae population tripled after three weeks. Dried algal tissue from 380 L tanks was extracted and used to determine neutral lipid accumulation at 6.64±0.08%. Fatty acid content was analyzed by GC- FID and found high amounts of unsaturated 18 carbon chains (18:2-18:4), more than previously shown. A comparison of oil from cells growing in 380 L and 1 L cultures showed culturing condition affected fatty acid profile. It was concluded from these data that Monoraphidium sp. Dek19 could be a useful phycoremediation tool of municipal wastewater in the upper Midwest. i NORTHERN ILLINOIS UNIVERISTY DEKALB, ILLINOIS MAY 2016 USE OF THE GREEN MICROALGA MONORAPHIDIUM SP. DEK19 TO REMEDIATE WASTEWATER: SALINITY STRESS AND SCALING TO MESOCOSM CULTURES BY ANTHONY ROBERT KEPHART ©2016 Anthony Robert Kephart A THESIS SUBMITTED TO THE GRADUATE SCHOOL IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE MASTER OF SCIENCE DEPARTMENT OF BIOLOGICAL SCIENCES Thesis Director: Gabriel P. Holbrook ii ACKNOWLEDGEMENTS I would like to thank many individuals for aiding me in the completion of my project. First and foremost, I would like to thank Dr. Gabriel Holbrook for accepting me into his lab and for all the support he has provided throughout my time at Northern Illinois University. I cannot fully express the depth of my gratitude for the opportunity he has given me. I would also like to thank my graduate committee members, Dr. W. Scott Grayburn and Dr. Neil Blackstone, for their all of their advice and review. I would like to thank my fellow graduate students, Nick Kirchner and Adam Hage, and the undergraduate researchers LeRoy Reinke and Josh Pierson, for their labor, advice, support, and friendship. I am also grateful for all of the outside personnel who aided my research including the members of the DeKalb Sanitary District, Lori Bross at the NIU Microscopy lab, Dr. Taesam Kim at the NIU Chem. Analytical lab, and Craig Shultz at the NIU PMBC greenhouse. I would like to thank the Department of Biological Sciences at Northern Illinois University for funding through a graduate assistantship. I also thank the Midwest ASPB and VentureWell for funding and presenting opportunities. Finally, I would like to express my gratitude to all of my friends and family for their advice and never ending emotional support. iii TABLE OF CONTENTS Page LIST OF TABLES ........................................................................................................................ vii LIST OF FIGURES ..................................................................................................................... viii Chapter 1. INTRODUCTION ...............................................................................................................1 Taxonomy, Morphology, and Habitat of Monoraphidium sp. Dek19 .....................1 Nutrient Requirements and Usage in Microalgae ....................................................4 Photosynthesis in Green Microalgae .......................................................................8 Lipid Synthesis in Microalgae .................................................................................9 Microalgae Global Distribution .............................................................................12 Water Treatment Regulations ................................................................................13 Water Treatment Processes at the DeKalb Sanitary District .................................15 Microalgae-Based Wastewater Treatment .............................................................16 Global Demand for Hydrocarbons and Renewable Energy ...................................17 Biofuel Sources ......................................................................................................18 Large Scale Culturing of Microalgae .....................................................................21 Chemical Stressors .................................................................................................22 Research Goals.......................................................................................................25 2. MATERIALS AND METHODS .......................................................................................26 Identification of Algal Species...............................................................................26 iv Chapter Page Storage of Microalgae Strains and Creation of Inoculum .....................................27 Midwestern Growth Conditions .............................................................................31 Effluent Collection .................................................................................................31 Culturing Conditions ..............................................................................................32 Basal Media Conditions ........................................................................................34 Absorbance Measured at 680 and 750 Nanometers ...............................................34 Cell Density ...........................................................................................................35 Cell Dry Weight .....................................................................................................35 Collection of Microalgae from Large Cultures ......................................................36 Absorption Spectra of Monoraphidium sp. Dek19 Pigments ................................37 Isolation of Monoraphidium sp. Dek19 via manipulation of growth conditions ...37 Nutrient Depletion .................................................................................................38 Nitrogen .....................................................................................................38 Phosphate ...................................................................................................39 Photosynthesis in Monoraphidium sp. Dek19 .......................................................40 Fluorescence Microscopy ......................................................................................41 Cell Imaging...............................................................................................41 Quantification with ImageJ ........................................................................42 Lipid Quantification and Analysis .........................................................................42 Colorimetric Method ..................................................................................42 Gravimetric Method ...................................................................................44 3. RESULTS ..........................................................................................................................47 v Chapter Page Growth Conditions at a Midwest Wastewater Treatment Facility.........................47 Temperature ...............................................................................................47