Metabolic capacities of anammox bacterium: Kuenenia stuttgartiensis Mariana Itzel Velasco Alvarez December 2014 Master’s dissertation submitted in partial fulfilment of the requirements for the joint degree of International Master of Science in Environmental Technology and Engineering an Erasmus Mundus Master Course jointly organized by UGent (Belgium), ICTP (Prague) and UNESCO‐IHE (the Netherlands) Academic year 2014 – 2015 Metabolic capacities of anammox bacterium: Kuenenia stuttgartiensis Host University: Radboud University, Nijmegen UNESCO-IHE Institute for Water Education Mariana Itzel Velasco Alvarez Promotor: Prof. dr. ir. Mike Jetten Co-promoter: Prof. dr. ir. Piet Lens This thesis was elaborated at Radboud University and defended at UNESCO-IHE Delft within the framework of the European Erasmus Mundus Programme “Erasmus Mundus International Master of Science in Environmental Technology and Engineering " (Course N° 2011-0172) © 2014 Nijmegen, Mariana Velasco, Ghent University, all rights reserved. CONFIDENTIALITY NOTICE – IMPORTANT – PLEASE READ FIRST This document may contain confidential information proprietary to the Radboud University. It is strictly forbidden to publish, cite or make public in any way this document or any part thereof without the express written permission by the Radboud Universit. Under no circumstance this document may be communicated to or put at the disposal of third parties; photocopying or duplicating it in any other way is strictly prohibited. Disregarding the confidential nature of this document may cause irremediable damage to the Radboud University. CONFIDENTIAL DO NOT COPY, DISTRIBUTE OR MAKE PUBLIC IN ANY WAY PLEASE CONTACT RADBOUD UNIVERSITY IF YOU RECEIVED THIS DOCUMENT IN ERROR. Acknowledgments Every success is never achieved alone; is part of a whole, where directly or indirectly is the input of people and situations. Therefore I feel grateful with all the people that were around me and made possible this project. This attainment was possible first with the support of my family. I want to thank my mother, Lourdes Alvarez Arroyo for always believing in me, even in the times when I did not believe in myself, for always encouraging, for all her love and companion, for always being there despite of the distance. To my little brother, Andrés Velasco Alvarez, for always cheering up hard moments with your sense of humour, and for remembering that life can always be simpler and easy going. To Fernando Munguia for being part of the family, a support and companion to the three of us. To the entire Microbiology department for the support, caring and offering so much knowledge and learning. To Lina Russ for her patience, enthusiasm and for being such a great supervisor, I could not have been luckier. To Mike Jetten and Huub Op den Camp for giving me the opportunity to form part of the research team, I learned more than I expected. To all my IMETE classmates (2012-2014), all of you became my second family during the master. Thank you for all the support, and the sharing, to Nadya, Gilda and Shilpi for all the adventures we shared. To Simon for his friendship and support. Even it might seem for granted I want to thank myself, for remaining healthy, and for not giving up at any challenge. To the incognito family that was present in all the countries that I visited and made my stay easier, for sharing happiness and peace under any circumstance. To the eternal lighthouse, the one that taught me how to smile and to know myself, with his never ending light. Summary Since the discovery of anammox bacteria in a wastewater treatment plant, several studies were developed for the better understanding of the characteristics that the bacteria possess. At first place the application of anammox bacteria promoted the deep study of their interactions and the ways their activity could be improved. Afterwards anammox bacteria were found to have an important role in the nitrogen cycle. This observation gave a step forward to the study of the global anammox presence in the natural ecosystems and their contribution to the loss of fixed nitrogen. However the complete biochemical mechanism of anaerobic ammonium oxidation has still some gaps which have been progressively elucidated through labeling experiments and dynamic population analysis. The aim of this thesis was to understand the interaction of anammox bacteria under ammonium limiting conditions, as it is found in the natural environment. With the enrichment of an alternative source of ammonium (amino acids), were investigated the processes that might be involved for anammox occurrence and the alternative pathways that they can adopt for the production of dinitrogen gas. The study comprehended the activity of the reactor system, labeling experiments and the phylogenetic analyses of the microbial population. In addition anammox bacteria is known to be found as non-pure culture system, which gives the opportunity for a coupling mechanism, such interactions can provide useful information for the application in the industrial field. List of Figures Figure 1. Nitrogen cycle (figure adapted from Van Niftrik & Jetten, 2012) Figure 2. Electron microscopy image of Candidatus Kuenenia stuttgartiensis cell (Van Niftrik et al., 2008) Figure 3. Phylogenetic tree based on 16S rRNA gene sequence (Schmid et al.,2005) Figure 4. Possible pathways for nitrate reduction (figure adapted from Kartal.,2008) Figure 5. Biochemical structure of glycine, glutamate and serine Figure 6. Glycine cleavage system (figure adapted from RAST,2014). Figure 7. Reaction of L-glutamate dehydrogenases (GDH) (figure adapted from RAST,2014). Figure 8. L-serine cleavage system, figure adapted from RAST 2014 Figure 9. Schematic diagram of the bioreactor Figure 10. NOx analyzer for nitrate set-up (figure adapted from: https://wiki.science.ru.nl ) Figure 11. Ammonium and nitrite concentration till it reached ammonium limitation Figure 12. Nitrite concentration during the first week (1a) and second week of the experiment (2a). Ammonium concentration during the first week (1b) and second week of the experiment (2b). Nitrate production during the first week (1c) and second week of the experiment (2c). Figure 13. Amino acids consumption during two days of the first week (1a,1b), amino acids consumption for two days during the second week of the experiment (2a,2b). Figure 14. Nitrite and ammonium concentration before amino acids enrichment Figure 15. Microbial growth monitoring before amino acids enrichment. Figure 16. Nitrate, nitrite and ammonium concentration of one week experiment. Figure 17. Reactor contamination with protozoa. Figure 18. Amino acids consumption of one week experiment in first day (1a), and third day (1b). Figure 19. Growth monitoring before amino acids enrichment. Figure 20. Nitrite consumption during the first week (1a), and second week experiment (2a). Ammonium concentration during the first week (1b), and second week of experiment (2b). Nitrate production in the first week (1c) and second week (2c). Figure 21. Amino acids consumption for two days enrichment day 2 (1a) and day 4 (1b). Figure 22. 15N-glycine enrichment in day2 (1a), and day 4 one week experiment (1b) Figure 23. 15N-glutamic acid enrichment in day 2(2a) and day 4 of experiment (2b) Figure 24. Nitrate and 15N-glutamate activity test (1), nitrate, 15N-glutamate activity test and nitrite (added at time=2) (2). Figure 25. Nitrate and 15N-glycine activity test (1), nitrate, 15N-glycine activity test and nitrite (added at time=2) (2). Figure 26. Monitoring of Kuenenia stuttgartiensis enrichment with amino acids (ser-glu-gly). One month stabilization under ammonium limitation (A),1 day of enrichment (B), 10 days of enrichment (C) after 4 days of no enrichment (D). Bar=10µm Figure 27. Monitoring of Kuenenia stuttgartiensis enrichment with amino acids (glu-gly). Reactor restarted with fresh biomass (E), 1 day of enrichment (F), 4 days of enrichment (G), and 14 days of enrichment (H). Bar=10µm Figure 28. Monitoring of Kuenenia stuttgartiensis enrichment with amino acids in the overall experiment. Enrichment with serine, glutamate and glycine after 10 days (I,J), enrichment with glutamate and glycine for 14 days (K), continuous enrichment with glycine and glutamate (L). Bar=10µm Figure 29. Maximum likelihood phylogenetic tree of bacteria present in the reactor system. Bootstrap values were calculated over 1000 repetitions. The sequences in the tree are noted with the code 16S.The bar shows 2% estimated sequence divergence. Figure 30. Maximum likelihood phylogenetic tree based on NirS protein sequences and the relationship with some nitrite reductase bacteria. Bootstrap values were calculated over 100 repetitions. The bar shows 50% of divergence estimated sequence. Figure 31. Maximum likelihood phylogenetic tree based on NirK protein sequences and the relationship with some bacteria containing copper nitrite reductase. Bootstrap values were calculated over 100 repetitions. The bar shows 50% of divergence estimated sequence. List of Tables Table 1. Anammox features Table 2. Free energy of Gibbs involved in nitrification, denitrification and anammox process Table 3. Anammox presence in natural ecosystems, ND No Data (Figure adapted from Hu et al., 2011) Table 4. Activity Test protocol Table 5. Probes used for FISH experiments Table 5. Primers for PCR amplification Table 6. Consumption rates of nitrite during enrichment at different days where it was observed activity. First enrichment with serine, glutamate and glycine (A), second