Michigan Technological University Digital Commons @ Michigan Tech Dissertations, Master's Theses and Master's Reports 2018 THE ABIOTIC STRESS RESPONSE OF HYDROPONIC VETIVER GRASS (Chrysopogon zizanioides L.) TO ACID MINE DRAINAGE AND ITS POTENTIAL FOR ENVIRONMENTAL REMEDIATION Jef Kiiskila Michigan Technological University, [email protected] Copyright 2018 Jef Kiiskila Recommended Citation Kiiskila, Jef, "THE ABIOTIC STRESS RESPONSE OF HYDROPONIC VETIVER GRASS (Chrysopogon zizanioides L.) TO ACID MINE DRAINAGE AND ITS POTENTIAL FOR ENVIRONMENTAL REMEDIATION", Open Access Dissertation, Michigan Technological University, 2018. https://doi.org/10.37099/mtu.dc.etdr/763 Follow this and additional works at: https://digitalcommons.mtu.edu/etdr Part of the Biochemistry Commons, Environmental Sciences Commons, and the Plant Biology Commons THE ABIOTIC STRESS RESPONSE OF HYDROPONIC VETIVER GRASS (Chrysopogon zizanioides L.) TO ACID MINE DRAINAGE AND ITS POTENTIAL FOR ENVIRONMENTAL REMEDIATION By Jeffrey D. Kiiskila A DISSERTATION Submitted in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY In Biochemistry and Molecular Biology MICHIGAN TECHNOLOGICAL UNIVERSITY 2018 © 2018 Jeffrey D. Kiiskila This dissertation has been approved in partial fulfillment of the requirements for the Degree of DOCTOR OF PHILOSOPHY in Biochemistry and Molecular Biology. Department of Biological Sciences Dissertation Advisor: Rupali Datta Committee Member: Dibyendu Sarkar Committee Member: Tarun K. Dam Committee Member: Victor Busov Department Chair: Chandrashekhar P. Joshi Dedication This dissertation is dedicated to my parents, David and Laurie Kiiskila, along with my grandmother, Nancy Kiiskila (1935-2017), for their unconditional love and support. Table of Contents List of figures .......................................................................................................................x List of tables ..................................................................................................................... xiv Author contribution statement ........................................................................................ xvii Acknowledgements .......................................................................................................... xix List of abbreviations ..........................................................................................................xx Abstract ......................................................................................................................... xxvii Introduction ..........................................................................................................................1 Occurrence of AMD in the US and the Tab-Simco site .............................................3 Floating Treatment Wetlands and Vetiver Grass ........................................................6 Combined Abiotic Stresses and Metabolomics ..........................................................9 Dissertation Objectives .............................................................................................10 References .................................................................................................................12 Chapter 1. A preliminary study to design a floating treatment wetland for remediating acid mine drainage-impacted water using vetiver grass (Chrysopogon zizanioides)a .......17 Abstract .....................................................................................................................19 Introduction ...............................................................................................................20 Materials and methods ..............................................................................................22 Hydroponic AMD phytoremediation experiment ..........................................22 Water chemistry and plant analysis ................................................................23 Results and discussion ..............................................................................................24 Effect of plants on water chemistry ................................................................24 Vetiver growth, metal uptake, and translocation ............................................29 Conclusions ...............................................................................................................36 Acknowledgements ...................................................................................................37 References .................................................................................................................37 Chapter 2. Remediation of Acid Mine Drainage-Impacted Water by Vetiver Grass: A Multiscale Long-Term Studya ............................................................................................41 Abstract .....................................................................................................................43 1. Introduction ....................................................................................................45 vii 2. Materials and methods ....................................................................................47 2.1. AMD collection and filtration ...........................................................47 2.2. Large-scale phytoremediation experiment ........................................48 2.3. Small-scale phytoremediation experiment ........................................49 2.4. Water sample characterization, metal removal, and toxicity screening of plant matter.................................................................................................50 2.5. Metal translocation and distribution analysis ...................................51 2.6. Statistical analysis .............................................................................51 3. Results and Discussion ...................................................................................52 3.1. Large-scale phytoremediation experiment ........................................52 3.1.1. Effectiveness of the vetiver rafts and plant health ..........53 2- 3.1.2. Effects of vetiver rafts on pH, conductivity, and SO4 removal 55 3.1.3. Metal removal by vetiver rafts and toxicity characterization .................................................................................56 3.1.4. Metal distribution and translocation throughout vetiver .64 3.2. Small-scale phytoremediation experiment ........................................65 2- 3.2.1. Effects of vetiver on pH, conductivity, and SO4 removal65 3.2.2. Metal removal by vetiver and translocation....................65 4. Discussion ......................................................................................................66 4.1. Effectiveness of the vetiver rafts and plant health ............................66 4.2. Effects of vetiver rafts and vetiver plants on pH, conductivity, and 2- SO4 removal .................................................................................................67 4.3. Metal removal by vetiver rafts and vetiver plants ............................68 4.4. Metal distribution and translocation throughout vetiver ...................69 Conclusions ...............................................................................................................71 Acknowledgements ...................................................................................................72 References .................................................................................................................72 Chapter 3. Metabolic Response of Vetiver Grass (Chrysopogon zizanioides) to Acid Mine Drainagea ............................................................................................................................78 Abstract .....................................................................................................................80 1. Introduction ....................................................................................................81 2. Materials and methods ....................................................................................82 2.1. Hydroponic Experiments and Sample Acquisition ...........................82 2.2. Water Characterization and Plant Physiological Response ..............82 2.3. Metabolite Extractions and LC-MS/MS ...........................................83 2.4. Data Processing and Statistical Analysis ..........................................83 3. Results and discussion ....................................................................................84 3.1. Physiological Response of Vetiver to AMD .....................................84 3.2. Metabolic Profiling of Vetiver Shoots in Response to AMD ...........88 viii 3.1.1 Upregulation of Amino Acid Metabolism and Downregulation of Proline................................................................88 3.1.1 Upregulation of Ornithine and Related Metabolites .......90 3.1.1 Upregulation of Sulfur-Containing Amino Acids and Glutathione Metabolism ...................................................................92 3.1.1 Upregulation of Cellular Respiration and Photosynthesis Metabolites........................................................................................93 3.1.1 Purine and Phospholipid Metabolism in Shoots .............94 3.3. Metabolic Profiling of Vetiver Roots in Response to AMD.............95 3.1.1 Phospholipid and Purine Metabolism in Roots ...............95 3.1.1