Nitrogen Removal and Lipid Production from Secondary Wastewater Using Green Alga Chlorella vulgaris By Zhouyang Liu B.S., Harbin Institute of Technology, 2007 Submitted in fulfillment of the requirements for the degree of Master of Science in Environmental Engineering in the Engineering & Applied Science College of the University of Cincinnati, 2012 Committee Members: Dr. Joo-Youp Lee (chair) Dr. Tim Keener Dr. Mingming Lu Abstract Increasing nitrogen discharges into natural water systems have caused more frequent eutrophication and other water quality issues. Microalgae are fast growing photosynthetic microorganisms that can assimilate nitrogen and phosphorus from water. Also, lipid content of certain strains of microalgae could reach over 60%, making microalgae excellent feedstock for biodiesel production. Green alga Chlorella vulgaris was tested for nitrogen removal and lipids production using secondary wastewater from municipal wastewater treatment plant. Around 60% of NH3-Nitrogen was removed after 48 hour, and removal rate was further increased to 75% when carbon dioxide gas was added periodically to control pH. When more active algae seeds were used, NH3-Nigrogen removal rate of 97.1% was achieved. Chlorella vulgaris was also very effective for removing low concentration of phosphate from secondary wastewater. When growing at normal conditions, Chlorella vulgaris contained more polar lipid than neutral lipid. Total lipid content of Chlorella vulgaris ranged from 10.6% to 14%, and fatty acids were mainly C16 and C18, making it good biodiesel stock. A freshwater microalgae survey in southwest Ohio was also conducted to provide useful information for future outdoor algae cultivation. 24 genera of cyanobacteria and 49 genera of eukaryotic algae were identified, with Synechococcus sp. and Cryptomonas sp. being the predominant prokaryotic and eukaryotic microalgae, respectively. ii iii Acknowledgments I would like to express my sincere gratitude to Dr. Joo-Youp Lee for his guidance and support throughout this study. I would like to thank Dr. Tim Keener and Dr. Mingming Lu for their helpful suggestions and feedbacks. I would also like to thank Dr. Daniel Oerther and Dr. Mau-Yi Wu for assisting and teaching me molecular biology techniques, and all the members in the lab especially Jinsoo Kim for their help with my experiments. Lastly, I want to thank my mother who never lost faith in me, and Zhen who was there with me when I was at the lowest point of my life, for which I am forever grateful. iv Table of Contents Chapter 1. Introduction ................................................................................................................... 1 1.1 Nitrogen and Phosphorus Pollution ...................................................................................... 1 1.2 Nutrients Removal Techniques ............................................................................................. 2 1.3 Nutrients Removal Using Microalgae .................................................................................. 3 1.4 Biofuels from Microalgae ..................................................................................................... 4 1.5 Objective of This Study ........................................................................................................ 6 Chapter 2: Molecular Survey of Freshwater Microalgae in Southwest Ohio Area ........................ 7 2.1 Introduction ........................................................................................................................... 7 2.2 Materials and Methods .......................................................................................................... 8 2.2.1 Sites Description and Sampling ..................................................................................... 8 2.2.2 Genomic DNA Extraction .............................................................................................11 2.2.3 PCR Amplification ........................................................................................................11 2.2.4 Cloning of rRNA Gene Fragments .............................................................................. 12 2.2.5 Sequence Analysis of Clones ....................................................................................... 13 2.3 Results and Discussions ...................................................................................................... 14 2.3.1 Cyanobacteria .............................................................................................................. 14 2.3.2 Eukaryotic Algae .......................................................................................................... 15 2.3.4 Comparison between Autumn and Winter Samples ..................................................... 18 2.4 Conclusions ......................................................................................................................... 22 v Chapter 3. Methods and Materials ................................................................................................ 24 3.1 Algae Cultivation ................................................................................................................ 24 3.2 Gravimetric Analysis of Lipids ........................................................................................... 25 3.2.1 Collection of Algae Biomass ....................................................................................... 25 3.2.2 Solvent Extraction of Total Lipids ............................................................................... 25 3.2.3 Silica Gel Purification .................................................................................................. 26 3.2.4 Esterification ................................................................................................................ 26 3.3 GC Analysis ........................................................................................................................ 26 3.4 Algae Cell Density Measurement ....................................................................................... 29 3.5 Ammonia Nitrogen and Phosphorus Analysis .................................................................... 29 3.5.1 Ammonia Nitrogen Measurement ................................................................................ 29 3.5.2 Orthophosphate Measurement ..................................................................................... 30 3.6 Total Inorganic Carbon Measurement ................................................................................. 30 Chapter 4. Ammonia Removal Using Chlorella vulgaris ............................................................. 31 4.1 Wastewater Properties ......................................................................................................... 31 4.2 Growth of Chlorella vulgaris in Closed Reactors .............................................................. 33 4.3 Nitrogen Removal Using Chlorella vulgaris ...................................................................... 36 4.3.1 Preparation of Algae Seeds .......................................................................................... 36 4.3.2 Experiment Set-up ....................................................................................................... 36 4.3.3 Nitrogen Removal Without Using CO2 ........................................................................ 37 vi 4.3.4 Nitrogen Removal with CO2 ........................................................................................ 41 Chapter 5. Lipid Analysis ............................................................................................................. 45 5.1 Lipids in Microalgae ........................................................................................................... 45 5.3 Lipid Extraction from Algae ............................................................................................... 47 5.2 Neutral and Total Lipids in Chlorella vulgaris ................................................................... 48 5.3 Polar Lipids in Chlorella vulgaris ...................................................................................... 52 Chapter 6. Conclusions ................................................................................................................. 56 References ..................................................................................................................................... 58 Appendix ....................................................................................................................................... 65 vii Chapter 1. Introduction 1.1 Nitrogen and Phosphorus Pollution Increasing nitrogen and phosphorus discharge into natural water systems poses great threat to water quality and human health. Excessive nutrients (nitrogen and phosphorus) in aquatic systems lead to more frequent eutrophication, which is a worldwide problem affecting both fresh and coastal marine water resources. In the US, point sources are responsible for more than 50% of the nutrients in rivers and streams in urban areas (Carpenter et al., 1998). Research shows that municipal wastewater treatment plants (WWTPs) account for about 75 percent of the nutrients from point sources (USGS, 1994; EEA 2005). Nitrogen and phosphorus are the main causes of surface water eutrophication. Freshwater eutrophication leads to excessive growth of phytoplankton, depletion of dissolved