PHYTOREMEDIATION OF HYDROCARBON-CONTAMINATED SOIL USING PHENOLIC-EXUDING HORTICULTURAL PLANTS A Thesis by ALEXANDRIA NYISHA IGWE Submitted to the Office of Graduate and Professional Studies of Texas A&M University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Chair of Committee, Arthur P. Schwab Co-Chair of Committee, Terry J. Gentry Committee Member, Raghupathy Karthikeyan Head of Department, David D. Baltensperger December 2015 Major Subject: Soil Science Copyright 2015 Alexandria Nyisha Igwe ABSTRACT A greenhouse experiment was designed to test phenolic-exuding horticultural plant species for their phytoremediation potential in soils contaminated with polycyclic aromatic hydrocarbons (PAHs). Species included high-phenolic-exuding plants: Malus sp., Osmanthus fragrans, Sambucus nigra, Castanea pumila, Morus alba, and Myrica cerifera and low-polyphenol-exuding plants: Ziziphus jujube, Ribes aureum, and Cassia fistula. The species were planted in soil amended with benzo[a]pyrene, phenanthrene, and pyrene. After 7 months, nitrogen, phosphorus, and potassium amendments were added each month for three months. Plant roots were harvested; polyphenols were ethanol- extracted and quantified using the Folin-Ciocalteu method. Rhizosphere DNA was extracted, quantified, and the 16S rRNA gene and ITS region were sequenced for bacteria and fungi, respectively. In addition, qPCR was conducted that targeted the 16S rRNA and ITS region of bacteria and fungi, respectively. The highest and lowest concentrations of phenolics were from Sambucus nigra and Ribes aureum, respectively. There were no significant differences between 16S rRNA and ITS abundance among treatments. Sequencing showed a significant difference between the rhizosphere bacterial community compositions on a global level. Specifically, several Actinobacteria were over-represented in the low-phenolic-exuding plants, while high-phenolic-exuding plants were over- represented by several Proteobacteria. There were no significant differences between the fungal populations of treatments and all were dominated by Ascomycota. However, high- phenolic exuders had a higher abundance of Basidiomycota, than the low-phenolic ii exuders. Results showed that different microbial communities were selected for by high polyphenol-exuding plants and low polyphenol-exuding plants. This selection was more pronounced in bacteria than in fungi. The drastic ways in which bacteria respond to phenolic inputs highlight their importance in phytoremediation. iii DEDICATION This work is dedicated to my Father in heaven and my mother on Earth. iv ACKNOWLEDGEMENTS I would like to thank my co-chairs, Dr. Terry J. Gentry and Dr. Arthur P. Schwab, for their guidance and support throughout the course of this research. Your mentorship, advice, and expertise were truly invaluable to me. I would like to thank my committee member, Dr. Raghupathy Karthikeyan, and substitute committee member, Dr. Robin Autenrieth for helping me finish strong. A special thanks goes to Dr. Pauline Wanjugi for teaching me all of the techniques I used to complete the microbial aspects of my research. I would also like to thank you for your mentorship and sound advice. I want to acknowledge Lauren Pitts, Ammani Kyanam, Keya Howard, Mariana Valdez, Dylan Laird, Nadezda Ojeda, the Black Graduate Students’ Association, the friends I made in College Station, Chardai Grays, Ngozi Okorafor and all my friends and family. My research would have progressed very slowly if it had not been for your friendship, moral support, and assistance. Finally, I would like to thank Texas A&M for their financial support. Thank you for considering me a high achieving scholar and investing in my future. v TABLE OF CONTENTS Page ABSTRACT .......................................................................................................................ii DEDICATION .................................................................................................................. iv ACKNOWLEDGEMENTS ............................................................................................... v TABLE OF CONTENTS .................................................................................................. vi LIST OF FIGURES ........................................................................................................ viii LIST OF TABLES ............................................................................................................ ix INTRODUCTION .............................................................................................................. 1 REMEDIATION OF PAHS ............................................................................................. 11 Biopiling/Composting .................................................................................................. 11 Landfarming ................................................................................................................. 13 Natural Attenuation ...................................................................................................... 13 Bioremediation ............................................................................................................. 14 Phytoremediation ......................................................................................................... 16 LITERATURE REVIEW: ROOT EXUDATES, MICROBIAL COMMUNITIES, AND PAH DISSIPATION ............................................................................................... 18 PURPOSE AND METHODOLOGY ............................................................................... 27 Purpose ......................................................................................................................... 27 Methodology ................................................................................................................ 28 Selection of plants .................................................................................................... 28 Soil ............................................................................................................................ 31 Sample collection and DNA extraction .................................................................... 31 Colorimetric phenolic assay ..................................................................................... 32 Community quantitative PCR .................................................................................. 34 Sequence analysis and community comparisons ...................................................... 37 RESULTS ......................................................................................................................... 39 Phenolic Concentration ................................................................................................ 39 vi Community Quantitative PCR ..................................................................................... 42 Community Composition, Diversity, and Estimated Richness .................................... 46 CONCLUSION ................................................................................................................ 62 REFERENCES ................................................................................................................. 67 vii LIST OF FIGURES Page Figure 1-Short-term and long-term health effects associated with polycyclic aromatic hydrocarbons ........................................................................................ 5 Figure 2-Bay-, L-, and K-region examples on polycyclic aromatic hydrocarbons ....................................................................................................... 5 Figure 3-Example of cancer causing structures in polycyclic aromatic hydrocarbons: dihydrodiol and K - region epoxide ............................................ 6 Figure 4-Phenolic concentration in gallic acid equivalents of the species used in this experiment ............................................................................................. 40 Figure 5-Rhizosphere 16S rRNA gene abundance of plants included in experiment ........................................................................................................ 44 Figure 6-Rhizosphere ITS abundance of plants included in experiment ......................... 45 Figure 7- Bacterial rhizosphere community composition of high- and low-phenolic exuding plants. ............................................................................ 50 Figure 8- Rhizosphere Proteobacteria class composition of high- and low-phenolic exuding plants. ............................................................................ 50 Figure 9-Phylogenetic tree of treatments based on rhizosphere bacterial community composition ................................................................................... 54 Figure 10-Principal Coordinate Analysis graph (PCoA) detailing how the treatment clustered based on rhizosphere bacterial community composition ....................................................................................................... 55 Figure 11-Fungal rhizosphere community composition of high- and low-phenolic exuding plants.. ........................................................................... 57 Figure 12-Phylogenetic tree of treatments based on fungal community composition ....................................................................................................... 60 Figure 13-Principal Coordinate Analysis