ANAEROBIC BIODEGRADATION OF POLYCYCLIC AROMATIC HYDROCARBONS USlNG FERRIC IRON AS TERMINAL ELECTRON ACCEPTOR by Kevin A. Robertson A thesis submitted to the Department of Chemical Engineering in conformity with the requirements for the degrce of Master of Science (Engineering) Queen's University Kingston, Ontario, Canada December, 1 998 copyright O Kevin A. Robertson, 1998 National Library Bibliothèque nationale du Canada Acquisitions and Acquisitions et Bibliographie Services services bibliographiques 395 Wdlington Street 395, rue Wellington OttawaON K I A W OttawaON K1AON4 Canada Canada The author has granted a non- L'auteur a accordé une licence non exclusive licence ailowing the exclusive permettant à la National Library of Canada to Biblothèque nationale du Canada de reproduce, han, distribute or sell reproduire, prêter7 distribuer ou copies of this thesis in microform, vendre des copies de cette thèse sous paper or electronic formats. la forme de microfiche/nlm, de reproduction sur papier ou sur format électronique. The author retains ownership of the L'auteur conserve la propriété du copyright in this thesis. Neither the droit d'auteur qui protège cette thèse. thesis nor substantial extracts fiom it Ni la thèse ni des extraits substantiels may be printed or otherwise de celle-ci ne doivent ê e im primés reproduced without the author's ou autrement reproduits sans son permission. autorisation. Abst ract Although aerobic biodegradation has been successfully demonstrated, a large niche exists whereby intrinsic bioremediation via ~e+r'e dudion would provide a significant economic advantage. This work examined a novel and promising in situ remed iation approach to address the widespread contamination of poly cycl ic aromatic hydrocarbons (PAHs) using ferric iron as the terminal electron acceptor. Two consortia enriched from contaminated soil/sediment, QU 1 and QU2, exhibited characteristics consistent with PAH degradation coupled to ferric iron reduction. In subsequent treatability studies, QU2 was shown to be capable of appreciable PAH mineralization of the low molecular weight compounds, such as naphthalene and phenanthrene. The poor solubility, and hence bioavailabi li ty, of the higher molecular weight compounds was probably the source of their recalcitrance, and so surfactants and cyclodextrin were investigated as aids to enhance their dissolution. At concentrations above their critical micelle concentration (CMC), Brij35 and Trition x-100 were both toxic to the microorganisms. Cyclodextrin did improve the rate and extent of phenanthrene mineralization slightly, but at higher cyclodextrin concentrations it appeared to be consumed as the preferent ial carbon source, thereby inh ib iting phenanthrene metabolism. The low solubility of ferric oxides is often ratecontrolli ng and so EDTA was used to chelate them. Although EDTA dramaticall y increased fenic oxide solubil ization, it only en hanced Fe'' reduction of monoaromatic hydrocarbons, and inhibited PAH (napthalene, phenanthrene, and anthracene) degradation. To detennine the effect of in situ conditions on naphthalene mineralization, the influence of various environmental factors was examined. The results showed that within a typical subsurface range of temperature (lO°C - 30°C), pH (6 - 8), and nutrient profile (nutrient rich, nutrient poor), minerali zation of PAHs could proceed. The capacity of the microorgani sms for hydrocarbon degradation coupled to iron reduction was a fundion of the bacterial population. When inoculated with QU 1, toluene mineralization occurred with a concomitant accumulation of ferrous iron. The mineralization of low and, to a lesser degree, high molecular weight PAHs catalyzed by QU2 was accompanied by signs consistent with the presence of an iron biogeochernical cycle. A mechanism was proposed whereby PAH rnetabolism occurred through a transfer of electrons to ferric oxide, resulting in the production of soluble Fe? Diffusion of the ferrous species into the overlying aqueous layer may have been followed by its re-oxidation to ferric iron which precipitated ont0 the water-sediment interface. The mechanism responsible for the regeneration is unclear, however several theories are presented. Co-Authorship The research and writing of this thesis was performed by Mr. Kevin Robertson. However, during the course of this project, several undergraduate students contributed work which went towards their senior theses. These students conducted experiments that were designed and supervised by Mr. Robertson, but their individual perspectives have provided a unique and valuable addition to this work. Their assistance cannot go without being recognized. Mr. Trevor Bugg examined the effects of synthetic surfactants on the bioremediation of PAHs. Figure 6.1 contains information related to work that he performed for his senior thesis in Engineering Chemistry. Mr. Nicolas Acay investigated the potential role of cyclodextrin in the remediation of PAH-contaminated sites. Figures 6.3, to 6.6 summarize some of this work, also for his senior thesis in Engineering Chemistry. Ms. Caroline Seto tested the selective ability of EDTA to solubilize iron forms of varying crystallinity. This data is included as Figure 7.2, and was part of her work for her senior thesis in Engineering Chemistry. Acknowledgements I owe many thanks to the numerous people who have offered their support along the way. I am especially grateful to rny supervisor, Dr. Juliana Ramsay, from whom 1 have grown in the field of biorernediation, and research, as well as in life. For her guidance, as well as her confidence in my abilities I am appreciative. Also, to Dr. Bruce Ramsay, who offered both insight and his home, I am grateful. I am truiy indebted to MarieCiaire Aly-Hassan at Ecole Polytechnique in Montreal for her many houn of dedication towards this project. She has made a valuable contribution and her efforts don't go unheeded. It has also been my pleasure to work alongside Janani Swamy, Todd Adamsson, and James Smith who offered encouragement and great friendships. 1 wish them the very best in their future endeavours. Departmental administration and Steve Hodgson found ways to help in almost any situation, and kept everything running smoothl y. Lastly to my friends, family, and new wife who extended great support under any circumstance, I give than ks. Table of Contents . . . . . ,.......................... . . ...... .................. Abstract ................... ... ..... .. ......................................................... Co-Authorship ............................. ............ ............................................. Acknowledgements ............................................................................................... List of Tables ................................. ........................................................... List of Figures Nomenclature ......................................................................................... . ............................ ....., ........ .... . Chapter 1 Introduction . .................... ........................................ Chapter 2 titerature Review .,,, ........................................................ PAHs as Priority Pohtants ........................ .. ........................... Entry into the Environment .......................................................................................... Fate ......... ............. . ................................................ Remediation ......................................................................... B ioremediation 2.5.1 Mass Transfer Limitations .......................................... 2.5.2 Chemical Structure Dependence ............................... 2.5.3 Substrate Mixtures .................................................... ................... ...... .... .......................................... Biostimulation .,., ................................ 2.6.1 PAH Bioavailability Enhancement 2.6.2 Nutrient Supplementation .......................... .. .............. .................... .............................. 2.6.3 Redox Conditions ,,, Anaerobic Microbial Degradation ........................ .. ................ ........... ............................................ 2.7.1 NitrateReduction .................................................... 2.7.2 SulphateReduction ............................. .. ......................... 2.7.3 Methanogenesis ......................................................................... lron Reduction 2.8.1 Ligand-Stimulated lron Reduction .............................. 2.8.2 Compounds Degraded .............................................. ................................................................... ............... 2.9 Summary Chapter 3 . Materiais and Methods .................... ..,., ..................................... .......................................................................... 3.1 Introduction 3.2 Ferric Oxide Synthesis .................... .... ............................. 3.3 lron Analysis ......................................................................... 3.4 14C Analysis .......................................................................... Chapter 4 . lnoculum Development ...................................... ................ ........ ............................................................. ......... 4.1 Introduction .............. ........ . .. .. ..................*........... 4.2 En richment Methods 4.2.1 Sources of lnoculum ................................................. 4.2.2 lnoculum Development ............................................ 4.2.3 Isolation .................................................................. 4.2.4 lnoculum Evaluation ................................................. .............................................................. 4.3 Results and Discussion .............................................................................. 4.4 Conclusions Chapter 5. Substrate Uti l ization ...................... .. ... ................................... 5.1 introduction .................................... ...................................... 5.2 Experimental ......................................................................... 5.3 Results and Discussion .......................................................... 5.3.1 Naphthalene ............................................................ 5.3.2 Phenanthrene ........................................................... 5.3.3 Toluene ................................................................... 5.3.4 PAH Family of Compounds ....................................... 5.4 Concl usions .......................................................................... . ......................................... Chapter 6 Enhancement of PAH Bioavailability .............................................................................. 6.1 Introduction ................... ... ..................................... 6.2 Synthetic Surfactants ............................................................... 6.2.1 Experimental ............................. ....,,.. ......... 6.2.2 Results and Discussion ................................... .. ...................................... 6.3 Cyclodextrin ............................................................... 6.3.1 Experimental 6.3.1.1 PAH Solubilization in an Aqueous Solution ... 6.3.1.2 PAH Desorption from a Sand Matrix .............. 6.3.1.3 Mobilization of PAHs through a Packed Column ................... .... ......................... 6.3.1.4 Biomineralization of PAHs ........................... 6.3 -2 Results and Discussion .................................... ............ 6.3.2.1 PAH Solubilization in an Aqueous Solution ... 6.3.2.2 PAH Desorption from a Sand Matrix .............. 6.3.2.3 Mobilization of PAHs through a Packed Column ...................................................... 6.3.2.4 Biomineralization of PAHs ........................... ................................. ,.............. ......................... 6.4 Conclusions Chapter 7 . iron Chelation .......................................... ........................ 7.1 Introduction ............... ................................. . ...... 7.2 Experimental ..................... .. ................................................. 7.2.1 Effect of EOTA on Fe Dissolution ............................. ., 7.2-2 Effect of EDTA on Phenanthrene Degradation ............. 7.3 Resultsand Discussion .......................................................... 7.3.1 Effect of EDTA on Fe Dissolution ............................... 7.3.2 EDTA-En hanced Hydrocarbon Oxidation .................... 7.4 Conclu sions ....................... .... ............................................... . Chapter 8 Environmental Factors ............................................................. introduction .......................................................................... Experim ental ......................................................................... Resu lt s and Discussion .......................................................... 8.3.1 Temperature ............................................................ 8.3.2 pH .......................................................................... 8.3.3 Ferric Oxide Crystal l inity, lnoculu m Percentage, Nutrients ................................................................. Concl usions ........................ .. .............................................. Chapter 9 . Redox State .......................................................................... Introduction ....................... ................................................. ,, Experi mental ............................. .. ........................................ Results and Discussion .......................................................... 9.3.1 Oxygen ................................................................... 9.3.2 Nitrate ..................................................................... 9.3.3 Sulphate .................................................................. 9.3.4 lron ......................................................................... 9.3.4.1 QU1 Consortium ......................................... 9.3.4.2 QU2 Consortium ......................................... 9.4 lmpii cations .......................................................................... . Chapter 10 Conclusions and Recommendations ...................................... References .................................................................................................... Curriculum Vitae .................................................... ................................ List of Tables . 2 i Physiochemical properties of selected PAHs relating to their bioavailability ................. ...... . .................................................... 12 2.2 Theoretical free energy change associated with the mineralization of PAHs coupled to various electron acceptors ..................................... 30 4.1 Growth medium and trace mineral compositions .............................. 40 4.2 Qualitative evaluation of plated rnicrobial species ............................ 43 4.3 PAH concentrations in QU2 consortium sediment ............................ 45 5.1 Experimental conditions used for spiking radio-labeled cornpounds .... 47 5.2 Cornparison of mineralization rates for organic substrates studied ....... 55