Evaluation of Sacred Lotus (Nelumbo nucifera Gaertn.) as an Alternative Crop for Phyto-remediation by Warner Steve Orozco Obando A dissertation submitted to the Graduate Faculty of Auburn University in partial fulfillment of the requirements for the Degree of Doctor of Philosophy Auburn, Alabama May 6, 2012 Keywords: Aquaponics, Heavy Metals, Constructed Wetlands, CWs Copyright 2012 by Warner Orozco Obando Approved by Kenneth M. Tilt, Chair, Professor of Horticulture Floyd M. Woods, Co-chair, Associate Professor of Horticulture Fenny Dane, Professor of Horticulture J. Raymond Kessler, Professor of Horticulture Jeff L. Sibley, Professor of Horticulture Wheeler G. Foshee III, Associate Professor of Horticulture Abstract Lotus, Nelumbo nucifera, offers a wide diversity of uses as ornamental, edible and medicinal plant. An opportunity for growing lotus as a crop in Alabama also has the potential for phyto-remediation. Lotus was evaluated for remediation of trace elements focusing on manganese (Mn), organic compounds targeting s-metolachlor and filtering aquaculture waste water. Lotus was evaluated for filtering trace elements by establishing a base line for tissue composition and evaluating lotus capacity to grow in solutions with high levels of Mn (0, 5, 10, 15, or 50 mg/L). Increasing Mn concentrations in solution induced a linear increase in lotus Mn leaf concentrations. Hyper-accumulation of Al and Fe was detected in the rhizomes, while Na hyper-accumulated in the petioles, all without visible signs of toxicity. Mn treatments applied to lotus affected chlorophyll content. For example, chlorophyll a content increased linearly over time while chlorophyll b decreased. Radical scavenging activity (DPPH) did not change over time but correlated with total phenols content, showing a linear decrease after 6 weeks of treatment. Ascorbic acid levels increased in response to lower Mn concentrations but at 50 mg/L or higher Mn treatment level, ascorbic acid levels were reduced. Lotus was evaluated for its potential as a phyto-remediator of organic compounds by growing plants in different concentrations of s-metolachlor. In seedlings, biomass accumulation was affected by the treatments. However, membrane integrity, respiration, and photosynthesis were not affected by the treatments. Mature plants did not show symptoms of toxicity but chlorophyll (Chl) changes in content were related to s-metolachlor concentrations (Chl a content ii increased while Chl b decreased). Potential s-metolachlor metabolites were found in rhizomes from treated seedlings. However, levels detected were too low to be identified. In addition, traces of s-metolachlor applied to seedlings were found in rhizomes, as well as, in leaves from treated mature plants. Lotus proved to be an effective phyto-remediator of nutrient + run-off (NH4 , NO3, P ) and suspended solids from waste water from intense aquaculture systems. iii Acknowledgments The author would like to express most sincere appreciation to Drs. Ken M. Tilt, Floyd M. Woods, Fenny Dane, Wheeler G. Foshee, J. Raymond Kessler, Jeff L. Sibley, Jesse A. Chappell, David J. Cline, Deacue Fields, and Juan Carlos Díaz for their guidance and wisdom through the pursuit of this degree. To my wife Monica Orozco Jenkins, for all her support and love during the process of this program. This work would have never reached conclusion without the blessing of Bernice Fischman whose wonderful personality and willingness to help kept me motivated and inspired. To Jo Anne Hall, who always believed and helped during my graduate academic career. Special thanks to John Olive, Arnold Caylor, George Griffith, and Grant Mitchell. To Bernice Barnette, Nancy Heard, and Tamikka Thomas who always were there when needed. The author is ever grateful to his mother María de los Angeles Orozco, his father Lusvin Mora Gutiérrez, his grandmother María Evangelina Obando Cardenas, his grandfather Francisco Orozco Muñoz, his aunts Sergia Orozco Muñoz, Gloria Orozco Obando, Lidia Orozco Obando, Marjorie Torres Villegas, his uncle Omar Antonio Mora Gutiérrez and to María H. Hidalgo, whose inspiration, confidence and prayers made this possible. To my brothers and sisters who always being there supporting me, I am grateful: Henry Orozco Obando, Luis Roberto Carmiol, Ronny Villegas, Dwight Mora Hidalgo, William Morgan Guevara, Walter Antonio Morgan Guevara, Ricardo Segura Amador, Miguel Antonio Matarrita Perez, Edwin Reyes, Patricia Rojas, Luis Flores Papuchin Capac, Viviana Leitón, María Ruth Martínez, Paula Biles, Kelly Billing, Nelly Jimenez, Victoria Ramírez, Roxana Ramírez, Kattia Mora Hidalgo, Josue Mora iv Laura, Steve and Bill Bancroft I want to thank you for your friendship, help and support. Finally, I know there is a whole group of people that I am not including; however, I really hope to have the opportunity to show you my appreciation. v Table of Contents Abstract ......................................................................................................................................... ii Acknowledgments........................................................................................................................ iv List of Tables ................................................................................................................................ x List of Figures ............................................................................................................................. xii Chapter 1: Review of Literature .................................................................................................. 1 1.1. Extension Programming for Introducing Lotus as a New Crop in Alabama ........... 1 1.1.2. Natural History of Lotus ........................................................................................ 2 1.1.2.1. Origin and Geographical Distribution of Lotus ........................................... 2 1.1.2.2. Biology of the Species ................................................................................. 4 1.1.2.3. Cultural Influences ....................................................................................... 6 1.1.2.4. Plant Uses ..................................................................................................... 7 1.1.2.4.1. Medicinal and Nutritional Values ...................................................... 8 1.1.3. Cultivation ........................................................................................................... 10 1.1.3.1. Sexual and Asexual Propagation ............................................................... 10 1.1.3.2. Soil Conditions ........................................................................................... 11 1.1.3.3. Fertilization ................................................................................................. 12 1.1.3.4. Container Management .............................................................................. 12 1.1.3.5. Pond Management ..................................................................................... 13 1.1.4. Vegetable Production .......................................................................................... 14 1.1.4.1. Field Production ......................................................................................... 14 vi 1.1.4.2. Planting and Harvest .............................................................................................. 15 1.1.4.3. Packaging and Postharvest Handling ......................................................... 16 1.1.4.4. Markets ...................................................................................................... 18 1.1.5. Cultivation of Lotus in Controlled Environments ............................................... 18 1.1.6. Evaluating How Plants Can Help to Clean up Polluted Environments ............... 19 1.1.6.1. Metolachlor as an Environmental Pollutant ................................................ 21 1.1.7. Phyto-remediation of Organic Compounds (S-metolachlor) ............................... 23 1.1.8. Phyto-remediation of Heavy Metals and Nutrient Run-off ................................. 24 1.2. Heavy Metal Effect on Antioxidant Capacity of Lotus Leaves ..................................... 26 1.3. Central Theme of Research ............................................................................................ 29 1.4. Literature Cited .............................................................................................................. 30 Chapter 2: Phyto-Remediation of S-Metolachlor Using Lotus (Nelumbo nucifera Gaertn.) ..... 38 2.1. Abstract .......................................................................................................................... 38 2.2. Introduction .................................................................................................................... 39 2.3. Materials and Methods ................................................................................................... 42 2.4. Results ............................................................................................................................ 50 2.5. Discussion ...................................................................................................................... 53 2.6. Literature Cited .............................................................................................................. 59 Chapter 3: Evaluation of Trace Metals Phyto-remediation Potential in Ornamental Lotus (Nelumbo nucifera