BIODEGRADATION OF A SULFUR-CONTAINING PAH, DIBENZOTHIOPHENE, BY A MIXED BACTERIAL COMMUNITY by Ellen M. Cooper Nicholas School of the Environment Duke University Date:_______________________ Approved: ___________________________ Dr. Heather Stapleton, Supervisor ___________________________ Dr. Andrew J. Schuler ___________________________ Dr. Richard T. Di Giulio ___________________________ Dr. Rytas Vilgalys ___________________________ Dr. Michael Aitken Dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Nicholas School of the Environment in the Graduate School of Duke University 2009 ABSTRACT BIODEGRADATION OF A SULFUR-CONTAINING PAH, DIBENZOTHIOPHENE, BY A MIXED BACTERIAL COMMUNITY by Ellen M. Cooper Nicholas School of the Environment Duke University Date:_______________________ Approved: ___________________________ Dr. Heather Stapleton, Supervisor ___________________________ Dr. Andrew J. Schuler ___________________________ Dr. Richard T. Di Giulio ___________________________ Dr. Rytas Vilgalys ___________________________ Dr. Michael Aitken An abstract of a dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Nicholas School of the Environment in the Graduate School of Duke University 2009 Copyright by Ellen M. Cooper 2009 ABSTRACT Dibenzothiophene (DBT) is a constituent of creosote and petroleum waste con- tamination, it is a model compound for more complex thiophenes, and its degradation by mixed microbial communities has received little attention. The chemical charac- teristics, environmental fate and ecotoxicology of DBT degradation products are not well understood. This research investigated DBT degradation in an enrichment culture derived from creosote-contaminated estuarian sediment using a suite of assays to moni- tor bacterial populations, bacterial growth, degradation products, DBT loss, and toxicity. Ultraviolet (UV) irradiation was evaluated as a sequential treatment following biodeg- radation. Additionally, to advance SYBR-Green qPCR methodology for characterizing mixed microbial communities, an alternative approach for evaluating qPCR data using a sigmoidal model to fit the amplification curve was compared to the conventional ap- proach in artificial mixed communities. The overall objective of this research was to gain a comprehensive understanding of the degradation of a model heterocyclic PAH, DBT, by a mixed microbial community, particularly within the context of remediation goals. DBT biodegradation was evaluated in laboratory scale cultures with and without pH control. The microbial community was monitored with 10 primer sets using SYBR- Green quantitative polymerase chain reaction (qPCR). Twenty-seven degradation prod- ucts were identified by gas chromatography and mass spectrometry (GC/MS). The di- versity of these products indicated that multiple pathways functioned in the community. DBT degradation appeared inhibited under acidic conditions. Toxicity to bioluminescent bacteria Vibrio fischeri more than doubled in the first few days of degradation, was never reduced below initial levels, and was attributed in part to one or more degradation products. UV treatment following biodegradation was explored using a monochromatic iv (254 nm) low-pressure UV lamp. While DBT was not extensively photooxidized, several biodegradation products were susceptible to UV treatment. At higher doses, UV treat- ment following DBT biodegradation exacerbated cardiac defects in Fundulus heteroclitus embryos, but slightly reduced toxicity to V. fischeri. This research provides a uniquely comprehensive view of the DBT degradation process, identifying bacterial populations previously unassociated with PAH biodegrada- tion, as well as potentially hazardous products that may form during biodegradation. Additionally, this research contributes to development of unconventional remediation strategies combining microbial degradation with subsequent UV treatment. v TaBLE OF CONTENTS Abstract..................... ........................................................................................................ iv List of Tables................ ....................................................................................................... x List of Figures....................................................................................................................xiii Acknowledgements ..........................................................................................................xxi Chapter 1. Introduction ..................................................................................................... 1 1.1. Research premise ............................................................................................ 1 1.2. Background .................................................................................................... 3 1.2.1. PAH contamination and environmental behavior ....................................... 3 1.2.2. Bioremediation of PAHs .............................................................................. 6 1.2.3. Degradation pathways and products .......................................................... 8 1.2.4. Monitoring bacterial populations by qPCR ............................................... 13 1.2.5. Photooxidation in contaminant reduction ................................................ 18 1.3. Research Plan ................................................................................................ 22 1.3.1. Hypotheses ...............................................................................................22 1.3.2. Research Objectives .................................................................................. 23 1.4. Scientific significance ..................................................................................... 25 Chapter 2. Microbial population dynamics during DBT degradation............................... 27 2.1. Introduction................................................................................................... 27 2.2. Methods ........................................................................................................ 30 2.2.1. Culture establishment, 16S rRNA gene clone library construction, isolation of pure cultures, and phylogenetic analysis ............................................ 30 2.2.2. qPCR primer design and assay ................................................................ 31 2.2.3. Degradation study ................................................................................... 34 2.2.4. Toxicity assay with bioluminescent bacteria. .......................................... 36 2.2.5. Statistical analyses. ................................................................................ 37 2.3. Results ........................................................................................................... 37 vi 2.3.1. Phylogenetic analysis .............................................................................. 37 2.3.2. DBT degradation, pH and microbial growth ............................................ 41 2.3.3. Population dynamics. .............................................................................. 46 2.4. Discussion ...................................................................................................... 50 2.5. Conclusion ..................................................................................................... 55 Chapter 3. Products of DBT degradation by a mixed microbial community .................... 56 3.1. Introduction................................................................................................... 56 3.2. Materials and methods ............................................................................... 62 3.2.1. Source of enrichment culture ................................................................. 62 3.2.2. DBT degradation studies ........................................................................... 62 3.2.3. Sample preparation and analysis by GC/MS ............................................. 63 3.2.4. Toxicity assay and dose-response experiments. ....................................... 69 3.2.5. Statistical Analyses .................................................................................... 70 3.3. Results ........................................................................................................... 71 3.3.1. Identification of DBT degradation products .............................................. 71 3.3.2. Toxicity of DBT and selected degradation products to V. fischeri ............. 89 3.3.3. Trends in toxicity during DBT degradation ................................................ 91 3.3.4. Trends in DBT and degradation products ................................................. 92 3.4. Discussion ...................................................................................................... 97 3.5. Conclusion ................................................................................................... 104 Chapter 4. Conventional and alternative approaches in the analysis bacterial popula- tions by SYBR-Green qPCR ................................................................................. 105 4.1. Introduction................................................................................................. 105 4.2. Materials and methods ............................................................................... 110 4.2.1.