Anaerobic ammonium oxidation in groundwater contaminated by fertilizers ELIF TEKIN Thesis Submitted to the Faculty of Graduate and Postdoctoral Studies University of Ottawa In partial fulfillment of the requirements for the M.Sc. degree in The Ottawa‐Carleton Geoscience Centre Friday September 28, 2012 Thèse soumise à la Faculté des études supérieures et postdoctorales Université d’Ottawa En vue de l’obtention de la maîtrise ès sciences L’Institut Ottawa‐Carleton de Géoscience Le vendredi 28 septembre, 2012 © Elif Tekin, Ottawa, Canada, 2013 Abstract Anaerobic ammonium oxidation (anammox) is a pathway that has been known for almost 2 decades, but few studies have investigated its importance in natural groundwaters. This thesis investigated the presence of anammox cells and the groundwater geochemistry of 2 sites (Elmira and Putnam) in southwestern Ontario where groundwaters are contaminated with high levels of nitrate and ammonium. Fluorescence in situ hybridization (FISH) was used to quantify the relative abundance of anammox cells in these waters. Our results showed that anammox cells could be detected in many wells at both sites and that their relative abundance varied between 0.45 and 4.81 % at the Putnam site, whereas it ranged between 0.8 to 8.4 % at the Elmira site. These values are within the same range as those obtained for marine and freshwater environments where anammox cells have been detected. In addition, indirect observations point to the fact that N cycling at the 2 sites might be linked to Fe and Mn reduction, but additional experiments are needed. In summary, our results corroborate the findings of N-labeled microcosm experiments which demonstrated that anammox was an important pathway of N cycling in those groundwaters and molecular analyses that detected important anammox organisms at the same sites. i Résumé L’oxydation anaérobique de l’ammonium (anaerobic ammonium oxidation (anammox) en anglais) est une réaction qui est connue depuis environ 20 ans, mais peu d’études se sont attardées à son importance dans les eaux souterraines. La présente thèse s’est intéressée à la présence des bactéries anammox et à la géochimie des eaux souterraines de 2 sites (Putnam et Elmira) contaminés au nitrate et à l’ammonium dans le sud-ouest de l’Ontario. La méthode FISH (fluorescence in situ hybridization) a été utilisée pour déterminer l’abondance relative de ces bactéries dans les eaux. Nos résultats indiquent que l’abondance relative des bactéries anammox varie de 0.45 à 4.81% au site Putnam, alors qu’elle varie entre 0.8 et 8.4% au site Elmira. Ces valeurs sont comparables à celles obtenues pour des environnements d’eaux douces et marines où les bactéries anammox ont été détectées. De plus, des observations indirectes indiquent que le cycle de l’azote aux 2 sites étudiés pourrait être lié à la réduction du Fe et du Mn, mais des expériences supplémentaires seront requises. En sommaire, nos résultats corroborent les travaux avec des de microcosmes contenant des composés d’azote isotopiques qui ont démontré que l’anammox est une réaction importante dans ces eaux souterraines, ainsi que des travaux moléculaires qui ont identifié plusieurs organismes capables d’oxyder de façon anaérobique l‘ammonium. ii Table of Content ABSTRACT I RÉSUMÉ II TABLE OF CONTENT III ACKNOWLEDGEMENTS V LIST OF TABLES VI LIST OF FIGURES VII 1. INTRODUCTION……………………………………………………. - 1 - 1.1 Nitrogen & Nitrogen reservoirs ………………………………………… - 1 - 1.2 Nitrogen as a contaminant………………………………………………. - 2 - 1.3 Nitrogen Cycling…………………………………………………………. - 4 - 1.4 Nitrogen fixation…………………………………………………………. - 6 - 1.5 Ammonia Assimilation (Immobilization) and Ammonification (Mineralization) ………………………………………………………………………… - 9 - 1.6 Nitrification………………………………………………………………. - 10 - 1.7 Denitrification……………………………………………………………. - 12 - 1.7.1 Dissimilatory nitrate reduction to ammonium………………………. - 15 - 1.7.2 Nitrate reduction coupled to iron oxidation…………………………. - 16 - 1.8 Anaerobic ammonium oxidation (Anammox)…………………………. - 16 - 1.8.1 Anammox bacteria……………………………………………………. - 20 - 2. OBJECTIVES AND HYPOTHESES…………………………………… - 21 - 3. METHODOLOGY……………………………………………………….. - 22 - 3.1 Study sites………………………………………………………………... - 22 - 3.1.1 Elmira, ON…………………………………………………………...... - 22 - iii 3.1.2. Putnam, ON……………………………………………………………... - 24 - 3.2 Field sampling methods…………………………………………………… - 25 - 3.3 Water analysis……………………………………………………………... - 27 - 3.3.1 On site field measurements……………………………………………... - 27 - 3.3.2 Groundwater geochemistry…………………………………………….. - 27 - 3.4 Fixation of cells and storage……………………………………………… - 28 - 3.5 Fluorescent in situ hybridization (FISH)………………………………… - 28 - 4. RESULTS…………………………………………………………………… - 30 - 4.1 Physico-chemical characteristics of the groundwater samples………… - 30 - 4.1.1. Putnam – Elmira Sampling Sites……………………………………… - 30 - 4.2 Bacterial abundance in ground waters using FISH……………………. - 43 - 4.2.1. Putnam…………………………………………………………………. - 43 - 4.2.2 Elmira…………………………………………………………………… - 45 - 4.3 Relationship between the water chemistry and the relative abundance of anammox cells………………………………………………………………… - 46 - 4.3.1 Putnam…………………………………………………………………... - 46 - 4.3.2 Elmira……………………………………………………………………. - 49 - 5. DISCUSSION………………………………………………………………. - 52 - 5.1 Anammox presence in contaminated groundwaters…………………..... - 52 - 5.2 Relationship between the presence of anammox cells and in situ aqueous geochemistry…………………………………………………………………… - 54 - 5.3 Improvements and future work………………………………………….. - 58 - 6. CONCLUSIONS……………………………………………………………. - 59 - REFERENCES………………………………………………………………… - 60 - iv APPENDIX……………………………………………………………………. 72 v Acknowledgements First, I would like to mention that I am sincerely thankful to my amazing supervisors Ian D. Clark and Danielle Fortin for showing me the biggest patience and giving me the biggest support. Needless to say none of this would have been possible without them. I would also like to thank Aquaterre for providing access to the Elmira site, Boran Kartal for his guidance and help with FISH and Yangping Xing for her guidance and advice on the project. Additional thanks go to Ben Moulton, Mudher Al Khafaji, Yves Moussallam, Michelle Chartrand, Jonathan C. Gapuz Mayo, Melanie Cousineau and Ivy Liu for being there for me at times when things seemed impossible, and to everyone in the G.G. Hatch laboratory, including Paul Middlestead and Patricia Wickham, for their help and support during those times we spent together with laughter and joy. I also thank Kerstin Brauneder, my closest person, for everything and my parents and family, especially my uncle Haldun Tezel and aunt Handan Tezel, for their biggest support. I am very lucky to have a great family like them. Finally, I would like to dedicate this work to my niece Eda whose soul and smile will be with me forever. This project was funded in full by a NSERC strategic grant led by I.D. Clark. vi List of Tables Page Table 1: Nitrogen reservoirs in all habitats. 2 Table 2: Nitrogen compounds as contaminants and different examples of 3 removal processes. Table 3: Gibbs free energy of several reactions that takes place in autotrophic 17 denitrification and energy yield difference between nitrite and nitrate. Table 4: Well numbers and depths at the Putnam and Elmira sites. 25 Table 5: Oligonucleotide probes used for FISH analyses. 27 28 Table 6: Fluorescence characteristics of fluorochromes under an epifluorescence microscope. vii List of Figures Page Figure 1: Overall view of the nitrogen cycle. 6 Figure 2: A proposed scheme of distal and proximal controls on 14 denitrification and denitrifiers Figure 3: Location of the Elmira site showing the fertilizer company with 23 the existing extraction wells and the chemical company. Figure 4: The Putnam Turkey manure site seen by satellite imaging. The 24 numbers refer to the well location Figure 5: Dissolved oxygen in the various wells at the Putnam site over the 30 course of 4 months Figure 6: Dissolved oxygen values at the various wells of the Elmira site 31 over 4 months of sampling. Figure 7: pH variations as a function of the sampling times and wells at 32 Putnam Figure 8: pH variation during the different months and in different wells at 33 the Elmira site. Figure 9: NO - concentrations (mg/L) at the Putnam site as a function of 34 3 wells and sampling times. Figure 10: NO - concentrations (mg/L) at the Elmira site as a function of 35 3 the wells and sampling dates. Figure 11: Nitrite concentrations at the Putnam site as a function of the 36 wells and sampling dates. Figure 12: Nitrite concentrations at the various Elmira groundwater wells 37 and sampling seasons. Figure 13: Ammonium concentrations in the various wells at Putnam 38 during the various samplings times. Figure 14: Ammonium concentrations in the various wells of the Elmira 39 site during the various samplings times. viii Figure 15: Fe and Mn concentrations as a function of the sampling time 40 and wells at the Putnam site. Figure 16: Fe and Mn concentrations as a function of the sampling time and wells 41 at the Elmira site. Figure 17: Relative abundance of anammox cells (with respect to total 42 bacterial counts) for all sampling sites and dates (a).Total abundance of anammox cells in the various Putnam wells for all sampling times (b). Figure 18: Relative abundance of anammox cells (with respect to total 43 bacterial counts) for all sampling sites and dates (a).Total abundance of anammox cells in the various Elmira wells for all sampling times (b). Figure 19: Relationship between the concentration of soluble nitrate in the 44 various wells of Putnam and the relative abundance of anammox cells. Figure 20: Relationship between the ammonium concentration in the 45 various wells of Putnam and the relative abundance of anammox cells. Figure 21: Relationship between the concentrations of dissolved Mn in the 45 various wells of Putnam and the relative abundance of anammox cells. Figure 22: Relationship between the concentrations of dissolved Fe in the 46 various wells of Putnam and the relative abundance of anammox cells. Figure 23: Relationship between the concentration of soluble nitrate in the 47 various wells of Elmira and the relative abundance of anammox cells. Figure 24: Relationship between the ammonium concentration in the 48 various wells of Elmira and the relative abundance of anammox cells. Figure 25: Relationship between the concentrations of dissolved Mn in the 49 various wells of Elmira and the relative abundance of anammox cells. Figure 26: Relationship between the concentrations of dissolved Fe in the 49 various wells of Elmira and the relative abundance of anammox cells. Figure A: FISH image obtained for the Putnam waters showing aggregates IX of cells and possible exopolysaccharides (EPS) ix