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Pattern of Microbial Degradation of Estrone and Triclosan Mixture and Its Effect on Soil Bacterial Community

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Pattern of Microbial Degradation of Estrone and Triclosan Mixture and Its Effect on Soil Bacterial Community Pattern of Microbial Degradation of Estrone and Triclosan Mixture and its Effect on Soil Bacterial Community by Ezinne Adabaram Osuji, B.S., M.B.A. A Thesis In Biological Science Submitted to the Graduate Faculty of Texas Tech University in Partial Fulfillment of the Requirements for the Degree of MASTER OF SCIENCE Approved Dr. Deborah L. Carr Chair of Committee Dr. John Zak Dr. Todd Anderson Mark Sheridan Dean of the Graduate School December, 2016 Copyright 2016, Ezinne Adabaram Osuji Texas Tech University, Ezinne Adabaram Osuji, December 2016 ACKNOWLEDGEMENTS The journey to writing my thesis would not have been possible without the help of some people who are worth mentioning. My sincere and deepest appreciation goes to my Advisor, Dr. Deborah Carr for giving me the opportunity to be a part of her team of graduate students, for believing in me, for being patient with me, always ready to share her wealth of knowledge with me all through the duration of the M.S program in Texas Tech University and while working on my thesis and for helping me improve not only as a student but as a biologist. I also appreciate the members of my committee; Dr. John Zak and Dr. Todd Anderson for their help, support, advice and research guidance. I am grateful to Dr. Rao Kottapalli and Dr. Pratibha Kottapalli of the Center for Biotechnology and Genomics, Texas Tech University, Lubbock Texas for their help and patience during the metagenomics sequencing and analysis of the data. The completion of this project would not have been possible without the assistance and willingness to help of all the other members of the D. Carr lab; Meijun Dong, Anisha Navlekar, Bigyan Rimal, and Jordan Brown for their time, help and inputs during the course of my research work both in the field and in the lab, especially Meijun Dong who was always willing to help with most part of the statistical analysis. I am also grateful to Texas Tech University Association of Biologist (TTUAB) for funding part of this research work. My deepest gratitude goes to my Parents, Dr. Sydney and Mrs Pauline Osuji, Brother, Chukwudi Osuji, Sister-in-law, Faith Osuji and Niece, Nwadiuto Osuji, for their support all through this program, for their unrelenting believe in me and encouragement even during difficult times and showing me that I can achieve anything in life once I am ii Texas Tech University, Ezinne Adabaram Osuji, December 2016 focused at it. I cannot thank my brother enough because without him I might not have been able to attend Graduate school. To my extended family here in Lubbock, in the United States and in Nigeria, thank you for all the encouragement all through this journey. Lastly, my sincere thanksgiving and worship goes to God Almighty for giving me the strength to go through this journey hale and hearty. Thank you Lord for your love towards me. iii Texas Tech University, Ezinne Adabaram Osuji, December 2016 TABLE OF CONTENTS ACKNOWLEDGEMENTS ......................................................................... ii ABSTRACT .................................................................................................. vi LIST OF TABLES ..................................................................................... viii LIST OF FIGURES ..................................................................................... ix LIST OF ABBREVIATIONS ....................................................................... x I. INTRODUCTION ..................................................................................... 1 Pharmaceutical and Personal Care Products (PPCPs) in the Environment .... 1 Impacts of Estrone in the Environment .......................................................... 2 Impacts of Triclosan in the Environment ....................................................... 4 Soil Microbial Community ............................................................................. 5 Research Goal ................................................................................................. 6 Hypotheses of the Study ................................................................................. 7 Specific Aims and Objectives ......................................................................... 8 II. MATERIALS AND METHODS ............................................................. 9 Description of Site .......................................................................................... 9 Soil Collection .............................................................................................. 10 Experimental Design .................................................................................... 10 Test Chemicals .............................................................................................. 11 Substrate Utilization profiling (Biolog Ecoplate) ......................................... 11 Statistical Data Analysis for Biolog Ecoplate .............................................. 13 High Performance Liquid Chromatography (HPLC) ................................... 14 Statistical Analysis for HPLC ....................................................................... 15 Metagenomics Analyses ............................................................................... 15 DNA extraction ............................................................................................. 16 Library Preparation and 16S rRNA gene sequencing .................................. 16 Bioinformatics Data Analysis ...................................................................... 18 iv Texas Tech University, Ezinne Adabaram Osuji, December 2016 Sequencing Data Processing .......................................................................... 18 Statistical Analysis of Sequencing data .......................................................... 19 III. RESULTS AND DISCUSSION ...........................................................20 Substrate Utilization Profiling analysis ........................................................ 20 Degradation of Test analytes ........................................................................ 22 16S Metagenomic analysis result ................................................................. 24 IV. CONCLUSION ......................................................................................31 Future Studies .............................................................................................. 32 LITERATURE CITED ...............................................................................33 FIGURES AND TABLES ...........................................................................43 v Texas Tech University, Ezinne Adabaram Osuji, December 2016 ABSTRACT Pharmaceutical and personal care products (PPCPs) associated with land farming of municipal wastewater effluent may potentially, persist in the soil and alter soil microbial community processes. Estrone (E1) and the anti-microbial agent, Triclosan, were examined for their potential to persist and disrupt soil microbial community function. Soil with 7 decades-long exposure to these chemicals (conditioned soil) and naive soil, which has not been previously exposed (unconditioned soil), was spiked with estrone, triclosan, or a 1:1 mixture of estrone: triclosan, and incubated for 90 days in the dark at 27°C. Control samples consisting of unspiked conditioned and unconditioned soil were included in the analysis. The community level physiological profile was examined using BIOLOG® EcoPlates™ for the ability of their microflora to utilize ecologically relevant carbon sources. There was a significant increase in substrate activity and substrate richness in all treatments. Principal component analysis (PCA) of the data showed the microbial community utilized different carbon substrates by day 90 whereas they had exhibited similar substrate utilization at day 0. Microbial degradation rates were compared over the 90 days incubation period using high performance liquid chromatography (HPLC). Estrone and Triclosan showed the same pattern of biological degradation in both conditioned and unconditioned soils. Half-lives were determined to range between 5.9-6.8 days for the estrone treatments and 24.1-26.9 days in the triclosan treatments. The rate of degradation of the estrone:triclosan mixture was the same as the individual compound. 16S metagenome analysis of the conditioned day 0 control soil and the conditioned day 90 control, E1, triclosan and the binary mixture of estrone and vi Texas Tech University, Ezinne Adabaram Osuji, December 2016 triclosan was done. The result showed that there was a decrease in species diversity between the control at day 0 and the other treatments at day 90, establishment of unique OTUs in each treatment group at day 90 and Bacillus sp. being the most dominant bacterium specie in all the day 90 treatments. vii Texas Tech University, Ezinne Adabaram Osuji, December 2016 LIST OF TABLES 1. Grouping of 31 carbon substrates in the Biolog Ecoplate into six different guilds. Adapted from Choi & Dobbs (1999) ........................................................ 56 2. Definition of parameters used in the biolog ecoplate analysis. ............................ 56 3. Mean utilization of carbon sources in all treatment groups at day 90 by optical densities (OD) read from the spectrophotometer after 168h of plate incubation .............................................................................................................. 57 4. Mean values of Shannon’s diversity (H), evenness (E) and substrate richness
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