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Impact of Tropical Plants on Microbial Activity and Diversity in Soil Contaminated with Petroleum Hydrocarbons

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Impact of Tropical Plants on Microbial Activity and Diversity in Soil Contaminated with Petroleum Hydrocarbons UNIVERSITY OF HAWAII UBRAR,( IMPACT OF TROPICAL PLANTS ON MICROBIAL ACTIVITY AND DIVERSITY IN SOIL CONTAMINATED WITH PETROLEUM HYDROCARBONS THESIS SUBMITIED TO THE GRADUATE DMSION OF THE UNIVERSITY OF HAW AI'I IN PARTIAL FULLFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE IN MICROBIOLOGY AUGUST 2006 By Alexandra Ku Shibata Thesis Committee: Francoise M. Robert, Chairperson Stuart Donachie Roger S. Fujioka ii We certify that we have read this thesis and that, in our opinion, it is satisfactory in scope and quality as a thesis for the degree of Master of Science in Microbiology. Chairperson iii ACKNOWLEDGEMENTS I sincerely thank my advisor, Dr. Francoise Robert, for her guidance throughout this project and my graduate experience. I regard her as not only a mentor but also a friend. I thank my committee members, Drs. Roger Fujioka and Stuart Donachie, for their time and expert comments. I also extend my appreciation to the individuals and organizations that provided assistance at different stages of this project: Dr. Tung Hoang, for his advice and continued support; Drs. Qingxiao Li and Traci Sylva, for allowing the use of their laboratory equipment; Renee Harada and Marisa Toma for their help with the methylene chloride extractions and GC analyses, respectively; and the U.S. Army Engineer Research and Development Center Environmental Laboratory and the U.S. Army Engineer District, Honolulu, HI, for procurement of the soil from Hickam Air Force Base. I am extremely grateful to my mother and sisters who, through their unconditional love and belief in me, continue to encourage me to achieve my goals. I especially acknowledge my husband, Terrence, for his patience and loving support throughout this journey. And I humbly thank God for being my strength when I needed it most and for blessing me every day of my life with the loving people around me. iv ABSTRACT The effect of plants (milo, oleander and buffelgrass) and a hexadecane and phenanthrene mixture (I g and 200 mg/kg soil, respectively) on the diversity and activity of hydrocarbon-degrading bacteria in a sandy coastal soil was investigated. Twolthird of hexadecane was degraded after 56 days. Hydrocarbon depletion was not plant-enhanced but was retarded slightly by milo and buffelgrass. Lipase activity, an a1kane-metabolism indicator, increased during rapid hexadecane depletion (days 0-56). The diversity of the dominant hexadecane-degrading bacteria was based on partial sequencing of 168 rDNA. On day 0, mainly Alphaproteobacteria were found By day 56, Gammaproteobacteria dominated the contaminated samples whereas similar numbers of Aiphaproteobacteria and Gammaproteobacteria genotypes dominated the uncontaminated samples. Alcanivorax was found in all contaminated samples except for buffelgrass rhizospheres, which harbored only Pseudomonas sp. IMT40. With little hexadecane left by day 114, similar abundances of Alphaproteobacteria and Gammaproteobacteria genotypes occurred in all samples. Alcanivorax had virtually disappeared. v TABLE OF CONTENTS ACKN"OWLEDGEMENTS •••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••• W ABSTRACT .................................................................................................................. Iv LIST OF TABLES••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••• x LIST OF FIGURES••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••• xll CHAPTER 1 •••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••• 1 INTRODUCTION ••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••• 1 1.1 Environmental pollution problem ............................................................... 1 I.l Composition of Petroleum Hyd."earbons••••••••••••••••••••••••••••••••••••••••••••••••••• 2 1.3 Bioremediation •••••••••••••••••••.••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••• 3 1.4 Phytoremed.iadon •••••••••••••••.••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••• 6 1.S Soil lipase activity as an Indicator of hydrocarbon degradation and overall activity of hydrocarbon-degrading microorganisms ••••••••••••••••••• 10 1.5.1 Use of soil enzymes to determine microbial activity .............................. 10 1.5.2 Role of lipase in microorganisms .......................................................... 12 1.5.3 Uptake and metabolism of alkanes ........................................................ 13 1.5.4 Link between growth on alkane-type of hydrocarbons and increased .... 14 lipase actiVIty m Dllcroorgamsms ....................................................... ... 1.5.5 Soi11ipase activity as an indicator of the progress ofbioremediation of petroleum hydrocarbon-contaminated soils ....................................... 17 1.6 Assessing microbial diversity using DNA-based techniques ••••••••••••••••••••• 18 1.6.1 peR amplification of the small subunit ofrRNA genes ......................... IS vi 1.6.2 Denaturing gradient gel electrophoresis (DGGE) of the variable 16S rDNA regions ....................................................................................... 20 1.7 Use of DGGE to analyze the distribution ofspeeifie funetIonal genes in microbial populations .........••...........•........•.............•................................. 13 1.7.1 Alkane hydroxylase system ................................................................... 25 1.7.2 Enzymes involved in the degradation ofphenanthrene .......................... 29 1.8 Scope of the project .................................................................................... 32 1.8.1 Relevance and findings from a previous phytoremediation mvesttgatton. ., ........................................................................................ 32 1.8.2 Goals and objeetives of this study ......................................................... 33 CHAPTER 2 .................................................... _...............•.........................................• 35 MATERIALS AND METHODS ........•...............................................••..•.•..••.••.••.•. 35 2.1 Experimental design ..................•.....•...................................•.....•..•............. 35 2.2 SoU preparation for bottom pots ............................................................... 38 1.3 Plant and soH preparation for top pots ..................................................... 39 1.4 Determination of plant biomass at harvest .•...•.........••.............................. 40 2.5 Hydrocarbon extraction from soH and analysis .....................•..•............... 41 2.5.1 Hydrocarbon extraction from soiL ....................................................... .41 2.5.2 Hydrocarbon analysis ............................................................................ 42 1.6 SoU Upase activity determination ....................................••..•...•..•....••..•....•. 43 2.6.1 Standard curve preparation. ................................................................... 43 2.6.2 Determination oflipase activity in soil samples .................................... .45 2.7 Enumeration of bacteria in soU and rhizospheres .................................... 46 vii 2.7.1 Heterotrophic bacteria ........................................................................... 47 2.7.2 Hexadecane-degrading bacteria ............................................................. 47 2.7.3 Phenanthrene-degrading bacteria. .......................................................... 48 2.8 Characterization of isolates of alkane and phenanthrene-degrading bacteria. ...........................................•..•....•.......•......•...........•...•.•................ 49 2.9 Genomic DNA extraction from isolates and cells from MPN wells .......... SO 2.10 PCR ampUfication ...................................................................................... 51 2.11 DGGE analysis ......................................................................................-. .... 55 2.12 Sequencing and computer analyses of partlall6S rRNA and alkB genes ........................................................................................................... 56 2.13 Statistical analyses •.....•.........•..••.•..•.........•............••.•.....•.•..•..•.••.•...•........... 57 CHAPTER 3 ................................................................................................................ 58 RESULTS AND DISCUSSION ........................................................................... 58 3.1 Degradation of petroleum hydrocarbon contaminants over time ............ S8 3.1.1 Hexadecane degradation ............................................. :......................... 58 3.1.2 Phenanthrene degradation ..................................................................... 60 3.2 Response of plants to hydrocarbon contaminant ...................................... 62 3.3 Response of bacterial populations to petroleum hydrocarbons and plant roots ................................................................................................. 68 3.3.1 Heterotrophic bacteria counts ................................................................ 69 3.3.2 Hexadecane-degrading bacteria counts .................................................. 72 3.3.3 Phenanthrene-degrading bacteria counts...............................................
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