Metabolic Capacities of Anammox Bacterium: Kuenenia Stuttgartiensis

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)

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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 enrichment with glutamate and glycine (B) and third enrichment with glutamate and glycine (C). Table 7. Consumption rates of amino acids during enrichment. First enrichment with serine, glutamate and glycine at day3 (A), second enrichment with glutamate and glycine at day3 (B) and third enrichment with glutamate and glycine at day4 (C).

List of Annexes

Annex 1. Fixation for fluorescence in situ hybridization (FISH)

Annex 2. High Pressure Liquid Chromatography system (HPLC) Annex 3. PCR amplification

Table of Contents

Chapter 1. Introduction 1

1.1 The nitrogen cycle 1 1.2 Physiology and biochemistry of anammox bacteria 3 1.3 Anammox in natural ecosystems 7 1.4 Anammox pathways 8 1.5 Research questions and objective 13

Chapter 2. Materials and Methods 14

2.1 Analytical methods 15 2.2 Amino acids enrichment 16 2.3 Activity test in batch 18 2.4 Microbial population analysis 19

Chapter 3. Results 21

3.1 Reactor activity 21 3.2 Enrichment with N-labeled amino acids in the reactor 30 3.3 Activity Tests with labeled amino acids 31 3.4 Fluorescence in situ hybridization (FISH) analyses 33 3.5 Phylogenetic tree for 16S 36 3.7 Phylogenetic tree for NirS 37 3.7 Phylogenetic tree for NirK 38

Chapter 4. Discussion 39

Chapter 5. Conclusion 44

References 45 Annexes 52 The author 58

Chapter 1. Introduction

1.1 The nitrogen cycle The nitrogen cycle is a combination of biological and abiotic processes of great importance for living organisms. Nitrogen is an element that is present in all living organisms and occurs in the environment in organic form as amino acids, proteins and DNA. It also constitutes 78% of the Earth´s atmosphere as dinitrogen gas (N2). However, despite the percentage of nitrogen present in the atmosphere it is still a limiting nutrient in primary production, because nitrogen gas is not accessible for plants and animals (Kuypers et al., 2003). The different forms of nitrogen found in the natural ecosystem are related to microbial activity in the whole cycle. Therefore during the last few years an increased effort has been put in the study and comprehension of the microorganisms that participate in the nitrogen cycle particularly in nitrogen gas production. For many years it was believed that only heterotrophic bacteria were able to convert nitrate into dinitrogen gas (denitrification) and to be the only important sink for fixed inorganic nitrogen (in the ocean). One of the most important discoveries of the last century is the anammox process (anaerobic ammonium oxidation), which converts ammonium and nitrite into dinitrogen gas (Mulder., 1992).

1 1. Nitrogen Fixation 2. Ammonification: mineralization of organic nitrogen 3. Nitrification: Aerobic ammonium oxidation (a) and nitrite oxidation (b) 2 4. Denitrification 4 5 5. Anammox process

Compound Formula Oxidation state 3a nitrogen − Nitrate NO3 +5 − Nitrite NO2 +3 Nitric oxide NO +2

Nitrous oxide N O +1 3b 2 Dinitrogen gas N2 0

Hydrazine N2H4 -2 + Ammonium NH4 -3

Amino Nitrogen R-NH2 -3

Figure1. Nitrogen cycle (figure adapted from Van Niftrik & Jetten,2012) Figure 1. Nitrogen cycle (figure adapted from Van Niftrik & Jetten, 2012)

The nitrogen cycle is described in Figure.1 addressing: nitrogen fixation, ammonification, nitrification, denitrification and anammox process. In the cycle the oxidation states of nitrogen can go from -3 to +5. Considering not only the inorganic but also organic nitrogen compounds, especially the amino acids in the mineralization process, which are of importance for this study and will be described further.

A. Nitrogen Fixation

N2 is the predominant reservoir for nitrogen on Earth. It requires great amounts of energy to break its bound because of its stable form. The process involves the reduction of N2 to NH3. Some microorganisms able to perform nitrogen fixation are Rhizobium sp. in symbiotic processes with leguminous plants, and a lot of species among the Clostridia under anaerobic conditions (Brock, 2006). B. Nitrification Nitrification is the process where ammonium is transformed to nitrite and then to nitrate; it occurs under aerobic conditions by ammonia-oxidizing bacteria (AOB) such as Nitrosomonas. The second step of nitrification for the formation of nitrite to nitrate is performed by nitrite oxidizing bacteria (NOB) such as Nitrobacter (Brock,2006). C. Denitrification Denitrification occurs under anaerobic conditions where nitrate is used as an electron acceptor and organic matter as electron donor, leading to the formation of dinitrogen gas (N2). This process is accomplished by several heterotrophic bacteria such as Bacillus sp., Paracoccus denitrificans, and Pseudomonas sp.. In addition to heterotrophic denitrification, autotrophic denitrification can also occur. Some bacteria such as Thiobacillus denitrificans and

Sulfurimonas denitrificans use sulphur compounds as electron donors with nitrate to form N2. Compared to heterotrophic denitrification this process is not dependent on organic matter or more precisely on organic carbon source for the formation of N2 (Knowles R. 1982). D. Anammox process Anammox process is the one to one combination of ammonium and nitrite to form dinitrogen gas. It occurs under anoxic conditions where ammonium acts an electron donor and nitrite as electron acceptor. It represents a shortcut in the formation of N2. Moreover the discovery of anammox process led to the application directly in wastewater treatment for nitrogen removal. Anammox bacteria do not require the addition of organic matter and aeration, becoming an alternative process for a more feasible and low cost operation. In comparison to the nitrification and denitrification process that is used commonly in wastewater treatment, where

2 the addition organic matter is required for denitrification and aeration for nitrification (Kuenen, 2008).

Anammox bacteria can be found in the marine ecosystem where they contribute significantly in the nitrogen loss (more than 50%) (Jetten et al.,2009). Since their discovery, several studies looked for the different habitats in which anammox could occur. The first study where anammox bacteria were related to the formation of dinitrogen gas in the environment was in the Black Sea which is the biggest anoxic basin of the world. Habitats in which anammox activity has been shown include tropical freshwater system (Schubert et al., 2006), and recently found in marine hypersaline sulphidic basins (Borin et al., 2013).

Until now some features and important characteristics of anammox bacteria have been investigated such as their physiology and metabolism. However there are still some gaps concerning their activity in the marine nitrogen cycle. In the natural ecosystems anammox bacteria have been found to occur under ammonium- limiting conditions (Jetten et al., 2009). Therefore the broad presence of anammox in the environment encourages exploring more about their activity, and more precisely for means of this study in the marine ecosystem.

1.2 Physiology and biochemistry of anammox bacteria The discovery of anammox dates to the end of the 20th century when a group of scientists observed the disappearance of ammonium in the presence of nitrate and the absence of oxygen in a denitrifying plant. Such observation promoted the study of such a reaction where it was suggested the use 15N-labelling experiments. In the presence of 15N-labelled ammonium 14,15 the production of N2was detected, instead of the already known product of canonical 15,15 denitrification N2. This observation confirmed the occurrence of an alternative pathway for ammonium oxidation, where hydroxylamine was not first formed as in aerobic ammonium oxidation process, but that another compound reacted with ammonium to produce hydrazine (an important intermediate for anammox process). In order to identify the responsible microorganism, the sludge from the reactor was enriched and cultivated in a sequencing fed-batch reactor. The further characterization of such microorganism was done by electron microscopy and PCR amplification of DNA, revealing the coccoid shape and their similarity with the Planctomycetes phylum (Kuenen, 2008). From these starting studies, several features of anammox bacteria have been found and are summarized in Table1. (Jetten et al., 2009):

Table1. Anammox features Phylum Planctomycetes Order Brocadiales Oxygen requirement obligate anaerobes

Carbon source CO2 Energy source Chemolithotrophic Doubling time 11 to 20 days Form Coccoid shape Size 800nmdiameter

Figure2. Electron microscopy image of Candidatus Kuenenia stuttgartiensiscell (Van Niftrik et al., 2008)

Anammox bacteria derive from a common ancestor within the phylum Planctomycetes which is perceived as a missing link between eukaryotes and prokaryotes (Strous et al., 2006). With 16S rRNA gene sequencing five anammox genera have been identified sharing a sequence similarity ranging from 87% to 99%. All anammox organisms belong to the same monophyletic order called Brocadiales although the large phylogenetic distance exists between them (Jetten et al.,2009).

Five genera of anammox bacteria that have been identified: 1. Kuenenia sp. 2. Brocadia sp. 3. Anammoxoglobus sp. 4. Jettenia sp. 5. Scalindua sp. The first four genera have been enriched from activated sludge or freshwater environments. The last one seems to be the only representative in the marine environment (Scalindua sp.).

Figure3. Phylogenetic tree based on 16S rRNA gene sequence (Schmid et al., 2005)

Anammox bacteria obtain their energy for growth from the combination of ammonium and nitrite and CO2, for biomass synthesis as a sole carbon source, making them autotrophs. The production of dinitrogen gas by anammox bacteria is given by the following equation (Strous et al., 1998):

+ − − + 1NH4 + 1.32NO2 + 0.066HCO3 + 0.13H − → 1.02N2 + 0.26NO3 + 0.066CH2O0.5N0.15 + 2.03H2O (1)

Anammox bacteria have a high affinity to their substrates, therefore nitrite and ammonium can occur in very low concentrations (Ks <5µM). On the other hand their metabolic activity is comparatively low about 15-80µmol of N2 formed per g dry weight of cells per min (Strous et al., 1998). The anammox metabolism implicates the formation of gaseous intermediates which are nitric oxide (NO) and hydrazine (N2H4) where the enzyme hydrazine synthase (HSZ) oxidizes − hydrazine into dinitrogen gas (See equation2&3). In the anammox pathway NO2 is not only used for N2 formation but also to release electrons for CO2 fixation, this hypothesis is still under study since it is possible that it might not require them under alternative conditions (Hu et al. ,in preparation). + + − NO + NH4 + 2H + 3e → N2H4 + H2O (2)

+ − N2H4 → N2 + 4H + 4e (3)

The energy yield from the anammox process of conversion of ammonium and nitrite to N2 is lower than compared to the canonical denitrification, which has a higher energy yield and therefore a higher growth rate (Jetten et al., 2009).

Table 2. Free energy of Gibbs involved in nitrification, denitrification and anammox process

Compared to other known prokaryotes, in anammox bacteria the energy generation takes place in a unique compartment called anammoxosome. The anammoxosome is surrounded by a unique bacterial membrane conformed by ladderane lipids (Van Niftrik et al., 2010). These unique ladderane lipids can also be used as biomarkers to trace anammox presence in the environment. Moreover in this compartment enzymes, such as cytochrome c proteins, hydroxylamine oxidoreductases (HAO) and the hydrazine synthase (HZS) can be found. These heme c proteins that are involved in anammox metabolism, give the characteristic red colour to anammox bacteria (Kartal et al., 2013).

Among the five anammox genera, the first genome published was the one of Kuenenia stuttgartiensis, which became available in 2006. The study of the genome revealed that the metabolic pathway they use for CO2 fixation is the acetyl-CoA pathway. Through experiments using stable carbon isotope trace marker 13C, the presence of labelled-C in the ladderane lipids was confirmed, which are as mentioned earlier important biomarkers for these bacteria (Kartal et al., 2013).

1.3 Anammox in natural ecosystems

Anammox bacteria have a great importance in marine nitrogen cycle. Since the loss of fixed nitrogen compounds in the ocean have been linked to anammox activity and to denitrification 6

(Dalsgaard et al., 2005). They occur at the aerobic-anaerobic interface, in the oceanic oxygen minimum zones (OMZ). Many studies have confirmed their presence in several habitats, such as the Black Sea, Lake Tanganyika, the Eastern Tropical South Pacific (ETSP) OMZ, and other ecosystems (See Table3). Their activity has been confirmed by 15N isotope labelling experiments (Dalsgaard et al., 2005). In the Black Sea for instance, anammox activity was shown by the decrease of ammonium concentrations in the suboxic zone with a depth above 97m (Kuypers et al., 2003). Also it was determined by the observation of the unique ladderane lipids that anammox bacteria possess (Kuypers et al., 2003).

Table 3. Anammox presence in natural ecosystems, ND No Data (Figure adapted from Hu et al., 2011)

Location Contribution (%) Reference

Marine sediments Equatorial Pacific ND Hong et al. 2011 Cascadia Basin (U.S.A) 40–42 Engström et al. 2009 Golfo Dulce (Costa Rica) ND Schmid et al., 2007 Gullmarsfjorden (Sweden) 23-47 Quan et al. 2008 Haiphong (Vietnam) <2 Amano et al. 2011 North Sea (North of the Friesian Front) ND Schmid et al. 2007 Randers Fjord (Denmark) 5–24 Risgaard-Petersen et al. 2004 South China Sea (China) ND Hong et al. 2011 Marine water columns Golfo Dulce (Costa Rica) 19–35 Dalsgaard et al. 2003 Black Sea 10–15 Kuypers et al. 2003 Namibian waters ~100 Kuypers et al. 2005 Freshwater ecosystems Lake Tanganyika (Kigoma) 9–13 Schubert et al. 2006 Groundwater (Canada) 18–36 Moore et al. 2011 Lake Kitaura (Japan) <40 Yoshinaga et al. 2011 Other ecosystems Hot springs (U.S.A.) ND Jaeschke et al. 2009 Hydrothermal vents (Mid-Atlantic Ridge) ND Byrne et al. 2009

Considering that anammox bacteria are slow growing microorganisms, it may be assumed that in the marine ecosystem it will be even slower, because of lack of nutrients and lower temperature conditions. However, even though anammox was discovered from a wastewater treatment plant where the optimal temperature is around 37°C, they have been also found to

7 occur in temperatures from -2°C to 43°C. In a more recent study in deep sea hydrothermal vents it was found their presence at 60 and 85°C (Jetten et al., 2009).

For the existence of anammox in the marine environment it is important to locate the origin of nitrite and ammonium in anoxic zones. Considering that in such areas there are low concentrations of ammonium, as a result of the fast consumption and recycling in water of organic matter, and the competition with ammonium oxidizers (Zehr & Kudela, 2011). Therefore anammox bacteria might be dependent on other biochemical processes to obtain their substrates. Some studies have determined the possible processes that might provide nitrite to anammox. Possible partners for the supply of nitrite are the ammonia-oxidizing bacteria or archaea or denitrifyers performing only nitrate reduction. For instance in the Black Sea the nitrification- anammox coupling with ammonia-oxidizing crenarchaea and γAOB has been confirmed. The activity of these microorganisms might supply nitrite to anammox in the suboxic zone (Lam et al., 2007). Moreover in the OMZ of Peru it was observed that nitrite production was a result also of ammonia oxidation by crenarchaeal amoA (ammonia monooxygenase gene subunit A) and the reduction of nitrate by possible nitrate-reducing α-protebacterium (Lam et al., 2008). The source of ammonium could be the degradation of organic matter or dissimilatory reduction of nitrate to ammonium (DNRA). The latter processes can also be performed by anammox bacteria itself (Kartal et al., 2007). However little is known about DNRA in the marine ecosystem and the possibility of another source of ammonium that could be available for anammox in order to inhabit such ecosystems (Jetten et al., 2009)

1.4 Anammox pathways Anammox bacteria generally live under ammonium limitation in natural ecosystems as mentioned earlier, therefore it is suggested that other biochemical processes provide the ammonium. One of the alternative processes that have been studied in anammox bacteria is DNRA in the presence of organic compounds such as acetate, propionate and formate (Strous 2006 & Kartal, 2007 ). For such studies, anammox activity was determined using 15N isotopes which is a reliable method to determine the activity and differentiate the production of N2 depending on the process. 30 The two-biochemical pathways to produce N2 formation are:

15 - 15 - 1. DNRA: NO3 can be first reduced to NO2 and afterwards to ammonium via + multiheme nitrite reductase (nrf) as identified for many enteric bacteria. Then the NH4 could 15 - 30 be combined with NO2 to form N2 by anammox bacteria. 15 - 15 - 15 15 2. Canonical denitrification transforms NO3 or NO2 to N N via nitrite, nitric oxide and nitrous oxide.

Figure 4. Possible pathways for nitrate reduction (figure adapted from Kartal., 2008).

15 - 15 14 As mentioned previously, the anammox process transforms NO2 to N N with unlabeled ammonium. On the other hand experiments with the model anammox bacterium K. 15 15 15 - stuttgartiensis showed that the production of N N is also possible from NO3 using organic acids as electron donors (Kartal, 2007). Indicating that anammox metabolism via - NO3 is carried out through DNRA. It consists of two steps: first the reduction of nitrate to nitrite and second the reduction of nitrite to ammonium. The pathway followed for the + - - production of NH4 involved NO2 , which was produced transiently from NO3 , indicating that - the reduction of NO3 is not performed as canonical denitrification via N2O. However in the study it was observed in small amounts the production of N2O which might come from the detoxification of NO which is one of the proposed intermediates of anammox reaction (Kartal et al., 2007).

In the marine environment nitrate is in abundance but there is limitation of organic electron - - donors. Consequently the limiting step in such reaction is the reduction of NO3 to NO2 as a result of electron donor competition with other microorganisms present. 15 15 15 - In conclusion anammox bacteria can also produce N N from NO3 while oxidizing an energy source similar to denitrification (Kartal et al., 2007).

Another possible source of ammonium next to DNRA is the presence of organic nitrogen in the marine ecosystem. Especially the presence of free amino acids would be a logical source of ammonium. The importance of amino acids in the global nitrogen cycle has been shown in several studies already fueling mostly denitrification (Zehr & Kudela, 2011). As a result it was drawn the attention to elucidate the role of free amino acids in the nitrogen cycle with special focus to the capability of anammox bacteria to use them as an alternative source of ammonium. Previous studies on marine rains coming from the precipitation in the Atlantic Ocean and in the Gulf of Mexico, showed the presence of dissolved free amino acids (DFAA) where glycine, alanine and serine appeared to be the most predominant. Such amino acids where all found with L configuration, indicating a biological origin (Mopper & Zika., 1987). Since the ocean is the main source for DFAA and primary amines, the recycling of nitrogen compounds occurs there. Recent studies using labelled 15N trace isotopes in batch experiments with Kuenenia stuttgartiensis, have shown the production of 15N14N, in the presence of labelled nitrate with 29 unlabeled glutamate. Therefore the production of N2 led to the question whether anammox might be using the amino group of the amino acids (14N) or if it might be provided from other source (Russ., personal communication). Further analysis are needed to conclude whether anammox is using the free amino acids as a source of nitrogen and carbon or whether the catabolism of DFAA occurs only coupled with another microorganisms. The possible mechanism of K. stuttgartiensis in amino acid degradation will follow a thorough analysis.

The use of organic electron donors is an attractive approach for anammox activity. More precisely under ammonium limiting conditions, where anammox bacteria may adopt alternative lifestyles for their survival. Thus it can be hypothesized that anammox bacteria might be able to use DFAA as a source of ammonium. This assumption is based on a genome analysis of these bacteria, where the presence of genes that code for the cleavage of certain amino acids was found. From genome analysis we would

10 conclude that K. stuttgartiensis cannot use all of the most abundant amino acids. However, presently there are enzymes that are not specifically identified or are called as hypothetical so the use of other amino acids cannot be excluded. Through the RAST (Rapid Annotation using Subsystem Technology,2014) database a closer look was taken at the possible enzymes that could cleave the amino acids: serine, glutamate and glycine.

Figure5. Biochemical structure of glycine, glutamate and serine (BIOC1010: Nitrogen Metabolism)

• Glycine cleavage system: Glycine is a non-essential amino acid. In the K. stuttgartiensis genome it is annotated as the system for glycine cleavage. The glycine-cleavage system is a multienzyme complex that catalyses the reversible oxidation of glycine, yielding carbon dioxide, ammonia, methylenetetrahydrofolate and a reduced pyridine nucleotide (NADH). The enzymes decarboxylase system (GDC) catalyzes oxidative decarboxylation and deamination of glycine + with the formation of CO2, NH3 and the concomitant reduction of NAD to NADH. The net reaction catalysed is:

Figure 6. Glycine cleavage system (figure adapted from RAST,2014).

• Glutamate cleavage by glutamate dehydrogenase: Three separate glutamate transport systems have been determined in Kuenenia genome. Of the three systems sodium/glutamate symport carrier protein (GltS) is specific for glutamate. Glutamate plays an important role between carbon and nitrogen metabolism in all living

11 organisms. For glutamate cleavage an enzyme is described in RAST database. The subsystem encodes only L-glutamate dehydrogenases (GDH). Such enzyme plays an important role for the synthesis of urea, where the ammonia produced is used in urea cycle and the 2- Oxoglutarate in the citric acid cycle.

Figure7. Reaction of L-glutamate dehydrogenases (GDH) (figure adapted from RAST, 2014).

• Serine cleavage: Serine is a non-essential amino acid. In plants and bacteria serine can be directly converted into cysteine (Cysteine formation route). Glycine catabolism proceeds mainly by interconversion with serine and requires the participation of tetrahydrofolate (THF) C1- transfer system. The major route for serine catabolism is the serine dehydratase reaction, leading to pyruvate (Pyruvate route). Another way of serine utilization is reconversion of it to 3-phosphoglycerate (Glycerate route). From what we know K. stuttgartiensis does not possess the enzyme catalysing this reaction.

Figure 8. L-serine cleavage system ( figure adapted from RAST, 2014).

Research questions and objective

Research questions:

 To what extent can anammox take up free amino acids under ammonium limited conditions?  Which amino acids are anammox bacteria able to use as a source of nitrogen which role do amino acids play in nitrogen gas formation?  Can this research have further applications in wastewater streams for simultaneous removal of nitrogen and carbon?

General objective

To test the capability of anammox bacteria to use free amino acids as a source of ammonium for the production of dinitrogen gas.

Specific Objectives

 To identify the possible pathways driven by anammox bacteria for dinitrogen gas formation.  To determine the activity of anammox and the possible coupling bacteria that contributes to form dinitrogen gas.

Chapter 2. Materials and Methods A continuously stirred tank reactor (CSTR) operated at 1.5l liter (working volume) with a submerged membrane, was installed for the enrichment and experiment development. The configuration of a submerged membrane bioreactor has similar characteristics as the conventional crossflow membrane bioreactor, with the difference that part of the biomass is recirculated using a recirculation pump, which is referred as bleed (van Haandel & van der Lubbe, 2010). The purpose of using membrane is to have longer retention time of the bacteria in the reactor, to compensate the slow growth of the bacteria. The reactor was inoculated with biomass from a reactor of Kuenenia stuttgartiensis which was not pure culture but predominantly present about 98% of the biomass.

Ar/Co2 Volume Bleed pump sensor pH sensor

Sampling Effluent pump Ar/Co DO 2 sensor

Base pump Bleed

Effluent

Base (KHCO3)

MBR

Medium Figure 9. Schematic diagram of the reactor operated

The reactor was continuously flushed with 10ml/min Air/CO2 (95/5%) to maintain the anoxic conditions (0% of O2) and to provide carbon source (CO2) to anammox bacteria, followed by an off-gas outlet to prevent overpressure in the system. The membrane was purchased as handmade because of small size requirements. The temperature and pH were maintained at 30°C and ~7.4 respectively. The fluxes in the reactor system were operated for a feed of 400mL/d, and an effluent of 360mL/d. With a retention time of 30 days which was attained by a bleed of 40mL/d. The reactor parameters such as temperature, pH, volume, dissolved oxygen (0%), agitation (125rpm) were controlled by Applikon in-Control system (Applikon Biotechnology, The Netherlands). The cultivation and enrichment of anammox bacteria followed the standard medium (Strous, et al. 1998) with the difference of the addition of ammonium in lower concentrations to obtain the ammonium limiting conditions. The standard flux of ammonium is of 4.8mM/d and 6.4mM/d of nitrite in a 1.5L volume reactor. The reactor was monitored till it reached a steady state in biomass density and ammonium limiting, according to the stoichiometry of anammox (Eq.4).

+ − − + − 1NH4 + ퟏ. ퟑퟐNO2 + 0.066HCO3 + 0.13H → 1.02N2 + 0.26NO3 + 0.066CH2O0.5N0.15 +

2.03H2O (4)

2.1 Analytical methods Ammonium was measured colorimetrically after 20 min of incubation in the dark. With 100µL sample (to an exetainer, productcode 728W, Labco Limited, high Wycombe) containing 40-400µM of ammonium and 2 ml of the tenfold diluted OPA Reagent. After incubation the fluorescence was read using the CARY Eclipse spectrofluorofotometer (Taylor et al. 1974). The method followed for nitrite was adapted for reading abosrbance in Photometry in Wallac 1420 VICTOR2™ Multilabel Counter instead of standard spectrophotometer. Nitrite was measured colorimetrically after 10 min of incubation at room temperature. The samples were prepared in a 96 wells plate with 50µL sample containing 5-100µM of nitrite, 50µL of reagent A (1% (w/v) sulfanilic acid in 1M HCl) and 50µL of reagent B (0.1% (w/v) Naphtylethylene diaminedihydrochloride (NED)). The fluorescence was read at 530 and 550nm, to have the average for 540nm absorbance (method modified for van Eck,1966).

Nitrate was measured by a gas-phase chemiluminescent reaction using NOA280i NOx analyzer, using a reagent (saturated solution of VCl3 in 1M HCl) that converts both nitrite and nitrate to nitric oxide at 95 °C:

- +3 + + 2NO3 + 3V + 2H2O --> 2NO + 3VO2 + 4H

A volume of 50µL sample was injected directly into the reagent for optimal conversion (see Figure6.), containing a concentration of 200 nM to 24µM. The analyzer detects nitrite and nitrate, therefore it was subtracted the nitrite measured to have the nitrate concentration.

Figure6. NOx analyzer for nitrate set-up (figure adapted from wiki.science.ru.nl,2014)

The amino acids were measured by High-performance liquid chromatography (HPLC), using FMOC-Cl, by derivatization of amino acids with an autosampler (Prostar 420). Samples were diluted with HCL 0.01M containing norvaline and the sample with a final concentration of 375pmol in 15µL, then dried in a 2mL HPLC-vial. The detailed procedure followed afterwards in HPLC equipment is described in Annex1.

2.2 Amino acids enrichment Three experiments were developed to test the metabolic capacity of anammox bacteria. The stock solution of amino acids was prepared for a total concentration of 600µM and sterilized with a Millipore filter of 0.2µm pore size. From the stock solution were added 8mL per day by pulse feeding. All the experiments were arranged to have approximately the same total concentration of amino acids (~640µM) in the reactor. The activity was monitored taking samples (1mL) every hour for eight hours from time=0 to time=8.

A. Test procedure with glycine, glutamate and serine The stock solution of amino acids was prepared for a total concentration of 120mM of serine, glycine and glutamate, having a concentration of 40mM of each of them. The absolute molarity of each amino acid in the reactor was of 248µM in 1500mL reactor final volume. The experiment was developed for 2 weeks. From the two weeks experiment the activity was followed for three days in a row of every week, having in total six days of monitoring.

B. Test procedure with glycine and glutamate (one week) In the second experiment only two amino acids were added glycine and glutamate, in order to 29 test the use of the amino group in the formation of N2. Thus glycine and glutamate were available as N-labelled amino acids. The absolute molarity of each amino acid in the reactor was of 320µM. The duration of the experiment was for one week, and the activity was followed for four days in a row.

C. Test procedure with glycine and glutamate (two weeks) The experiment with glycine and glutamate was repeated with fresh biomass of Kuenenia stuttgartiensis. The time period of the experiment was for two weeks. The stock solution of amino acids was the same as the previous experiment with glycine and glutamate. The activity was monitored from the first day of experiment and two days in a row for every week, having in total 5 days measured.

Trace experiments with N- labeled glutamate and glycine A stock solution of labelled-N amino acids was prepared in a total concentration of 120mM. The experiment was performed in batch conditions, with no escape of the gas produced and no feed for a period of 8 hours. The gas was measured every hour to identify the dinitrogen 29 30 gas produced by anammox process ( N2), denitrification and the possibility of DNRA ( N2). The first experiment was performed adding approximately a concentration of 640µM of 15N- glycine by pulse feeding to the reactor. In the second experiment, were added 320 µM of non-labeled glycine and 320µM of 15N- glutamic acid.

For the N-labelled experiments, nitrogen ions were measured by Gas Chromatograph- Mass Spectrophotometer (GC-MS), injecting 50µL sample of the headspace in the GC-MS (Agilent 5975C inert MSD).

Bacterial Growth monitoring The growth of Kuenenia was monitored by OD (Optical Density) on a daily basis. It was measured in a spectrophotometer (Bio-Rad, USA) at 600nm. However since this method gives a rough estimate, protein concentrations were also measured in all the pellets from the samples taken. The protein assay was done by Bio-Rad protein assay, based on the method of Bradford using Immunoglobulin G (IgG) as a standard. The pellets were incubated at 90°C, for half an hour with 1 mL of NaOH 1M, and then neutralized with 1mL of 1M HCl. After extraction, the protein was measured colorimetrically at 595nm after 5min of incubation, 800µl from the (diluted) sample containing 1-20µg of protein and 200µl of the reagent.

2.3 Activity test in batch The experiments were developed in 10mL serum bottles with 5mL of biomass, harvested from the reactor under ammonium limitation. The bottles were sealed with rubber stoppers, and made anoxic conditions by applying alternatively under-pressure and Argon gas. An overpressure of 1 bar was maintained in the bottles. All the bottles were shaken continuously (100rpm) and maintained in a temperature of 30°C. The dinitrogen gas was measured every hour in every bottle, taking a volume of 50µL from the headspace.

Table 4. Activity Test protocol

mM 15 + - - 15 15 Bottle Test NH 4 NO2 NO 3 N-glycine N-glutamate 1 Positive Control 0.5 0.5 2 5 2 Labelled Glutamate 2 5 3 Labelled Glutamate 0.25* 2 5 4 Labelled Glutamate 0.25* 1 Positive Control 0.5 0.5 2 Labelled Glycine 2 5 3 Labelled Glycine 0.25* 2 5 4 Labelled Glycine 0.25* 2 5 * Added after 2hrs

2.4 Microbial population analysis

In any biological experiment is important to have an overview of the microbial population. As a non-pure culture system is important to identify any changes on the population diversity.

Fluorescence in situ Hybridization (FISH) FISH will provide qualitative and quantitative information about the bacteria community in the reactor and the possibility of a co-culture activity. Samples for FISH were taken from the reactor from the biomass (1mL). First analysis was from day 1 of enrichment, day 10 of enrichment, after 4 days of no enrichment. Second analysis was from day 1 of enrichment, 4 days of enrichment, and 14 days of enrichment. Third analysis was from 10 days of and for 14 days enrichment. The 1mL samples were fixed in paraformaldehyde and stored at -8°C till their analysis. FISH analyses were done in three phases following the activity from the enrichment procedure, described previously. The probes are purchased as AMX820Cy3, Cy5 and 5(6)- carboxyfluoresecein-N-hydrosuccinimide ester (FLUOS) labeled derivatives from Thermo Electron Corporation (Ulm,Germany). The probes used were AMX820Cy5 (blue) targeting anammox BETA420Cy3 (red) for targeting β-proteobacteria (Kartal., 2008).

Table 5. Probes used for FISH experiments

Probe name Target organism(s) Formamide Colour Sequence (from 5' to 3') References AMX-0820 Anammox 30-40 AAA ACC CCT CTA CTT AGT GCC C Schmid et al., 2001 EUB mix most Bacteria 0-80 GCT GCC TCC CGT AGG AGT Amann et al., 1990 BET-42a # ß-proteobacteria 35 GCC TTC CCA CTT CGT TT Manz et al., 1992 ALF-0968 a-proteobacteria 20 GGT AAG GTT CTG CGC GTT

Clone Library A. DNA isolation: 1mL sample was taken from the reactor on the 10th of June. DNA was isolated using the Power Soil kit (DNA) (MO BIO, USA) following the manufacturers instuctions. B. PCR amplification: Polymerase Chain Reaction (PCR) consisted on analysis on the identification of 16S genes and the presence of enzymes that are specific for denitrifiers NirS and NirK. The forward and reverse primers were selected from the probe list to target bacteria and the enzymes.

Table 6. Primers for PCR amplification Target gene Primer name Sequence Reference 16S 616F AGAGTTTGATYMTGGCTCAG Juretschko et al. 1998 16S 630R CAKAAAGGAGGTGATCC Juretschko et al. 1998 nirS cd3aF GTSAACGTSAAGGARACSGG Michotey et al. 2000 AEM nirS R3cd GASTTCGGRTGSGTCTTGA Throbäck et al. 2004 FEMS ME nirK F1aCu ATCATGGTSCTGCCGCG Hallin et al. 1999 AEM nirK R3Cu GCCTCGATCAGRTTGTGGTT Hallin et al. 1999 AEM

The reaction mixtures with the samples consisted of 1µL sample, 0.5µL of each primer, 10.5µL water and 12.5 PCR Quanta mix (Quanta BioSciences, Inc. USA), having a total volume of 25µL. The 16S amplification consisted in a thermal cycling with an initial denaturation of the DNA at 94°C for 5 min, followed by 40 cycles of denaturation at 94°C for 45 s, annealing at 52°C for 1min, and elongation at 72°C for 1min, the cycling was completed by a final elongation of 72 °C for 1min (Juretschko et al., 1998). The nirS and nirK fragments were amplified with an initial denaturation of the DNA at 94°C for 5 min, followed by 30 cycles of denaturation at 94°C for 1 min, annealing at 50°C for 45s, and elongation at 72°C for 45s, the cycling was completed by a final elongation of 72 °C for 7min (Throbäck et al., 2004). For nirK it was repeated the cycling with a gradient temperature for an optimal annelaing of 39 to 55.8°C. Positive controls containing Scalindua were included as well as negative control with no addition of DNA.

C. Ligation and plasmid isolation: PCR products, were ligated introducing the vector pGEM®-T Easy Vector (Promega, USA). Then the plasmids were cloned using E. coli. Colonies were chosen after cultivation according to the blue/white screening technique, eight colonies with the insert (white colonies) were chosen. Afterwards the plasmids were isolated using the GeneJET Plasmid Miniprep Kit (Thermo Fisher Scientific Inc.) and then sent for sequencing to a private laboratory (BASECLEAR BV, Leiden, Netherlands).

D. Reference database construction: With the results of the sequencing it was constructed the phylogenetic tree, using the Genbank (NCBI,2014). Each sequence was blasted to find the closest relative. Then the sequences were aligned to construct the library using the software Molecular Genetic Analysis version 6 (MEGA.6).

Chapter 3. Results In this chapter the results will be described in three parts, with the same order as the methodology description. First with the activity of the reactor, second part comprehends activity test with 15N-labelled experiments and finally the phylogenetic analyses.

3.1 Reactor activity The activity of the reactor in the presence of amino acids was determined in each experiment based on nitrite, nitrate and amino acids consumption. A brief overview of the results - obtained is shown in Table 6 and 7. In the experiments A and C a similar value of 112 NO2 µmol·mg protein-1 h-1 (3 times more than the initial) and 118.12 (4 times more than the initial) was reached respectively. While in the experiment B the activity decreased due to contamination. The amino acids consumption did not have a significant difference in the three experiments. Regarding the affinity for a certain amino acid is not representative since the initial concentration of amino acids was not exactly the same in the three experiments.

Table 7. Consumption rates of nitrite during enrichment at different days where it was observed activity. First enrichment with serine, glutamate and glycine (A), second enrichment with glutamate and glycine (B) and third enrichment with glutamate and glycine (C).

- Activity µmol NO2 /mg protein h

Day Experiment A B C 2 37.70 67.55 3 112.04 26.12 4 40.20 70.06

5 118.12

Table 8. Consumption rates of amino acids during enrichment. First enrichment with serine, glutamate and glycine at day3 (A), second enrichment with glutamate and glycine at day3 (B) and third enrichment with glutamate and glycine at day4 (C). Activity µmol amino acid /mg protein·h Experiment Serine Glutamate Glycine A 218.35 494.15 226.97 B - 376.86 397.80 C - 259.67 251.86

A. Enrichment with serine, glutamate and glycine In the first place the reactor was monitored and adapted to reach ammonium limiting conditions.

2 1,8

1,6

1,4

(mM)

- 1,2 2 Ammonium 1 Nitrite

or NOor 0,8

+ 4 0,6

NH 0,4 0,2 0 0 5 10 15 20 25 30 Time (days)

Figure 11. Ammonium and nitrite concentration till it reached ammonium limitation

The graph shows the start of operation (day 1) till it reached ammonium-limiting conditions (day 28). The progress to reach ammonium-limiting conditions was not straightforward; we needed to test different ratios of ammonium to nitrite till residual nitrite appeared. For the standard activity of anammox bacteria, the concentration in the influent of ammonium was of 4.8mM/d of ammonium. In order to reach the ammonium-limiting conditions the concentration of ammonium was decreased, first to 4.53mM/d with a ratio of 1.41 of nitrite, with no residual nitrite. Then it was decreased to 4.4mM/d with a ratio of 1.45 of nitrite, which resulted in 0.94mM of residual nitrite and no ammonium. Therefore ammonium + - limitation in the reactor was attained with 4.4mM NH4 /d and 6.4mM NO2 /d (Eq.5).

+ − − + − − 1NH4 + ퟏ. ퟒퟓNO2 + 0.066HCO3 + 0.13H → 1.02N2 + 0.26NO3 + ퟎ. ퟏퟕNO2 (5)

When the biomass concentration was maintained at 0.5 to 0.6 of optical density at 600nm

(OD600) and ammonium limiting conditions, the enrichment with amino acids was performed. The first experiment was developed for 2 weeks, adding serine, glutamate and glycine in the same concentration (248µM) for 14 days. The activity was followed by taking 1 mL sample

22 of the reactor for 8hours during three days in a row every week. The values measured are shown from starting point (time=0) till the last point measured (time=7).

1a 2a 0.25 0.5 0.45 0.2 day 1 0.4 day 10 day 2 0.35 0.15 0.3 day 11 day3 0.25 day 12

(mM) 0.1 0.2 0.15 0.05 0.1 0.05 0 0 0 2 4 6 8 0 2 4 6 8 Time (hours) Time (hours) 1b 2b 1 1 0.9 0.9 0.8 0.8 0.7 0.7 day 1 day 10 0.6 0.6 day 2 day 11 0.5 0.5 day3 day 12 (mM) 0.4 0.4 0.3 0.3 0.2 0.2 0.1 0.1 0 0 0 2 4 6 8 0 2 4 6 8 Time (hours) Time (hours) 1c 2c 3 1 2.5 0.9 0.8 2 0.7 day 1 0.6 1.5 day 10 day 2 0.5

(mM) day 11 1 day3 0.4 day 12 0.3 0.5 0.2 0.1 0 0 0 2 4 6 8 0 2 4 6 8 Time (hours) Time (hours)

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).

At day 1 of adding amino acids the residual nitrite was maintained at a concentration of 0.23 ± 0.004 mM. At day 2 at time=0 there is little residual nitrite around 40µM and after that time it remains at low concentrations (<30µM). By day 3 at time=0 there was also residual nitrite but in higher concentrations (0.2mM) and after adding the amino acids there was a significant decrease of nitrite concentration to 4µM after 2 hours. These results gave an indication that there might be ammonium available for anammox activity and therefore residual nitrite was

consumed. It is important to notice that the fact of having higher residual nitrite at time=0 in day 3 compared to day 2, could possibly be because of lag phase in activity during the night which might not be visible in the results. During the first week no ammonium was present indicating the completely consumption of it. Figure8. Ammonium concentration over time However in the second week, ammonium started to accumulate reaching a concentration of 0.8mM on day12. Considering that the amino group of the amino acids would be released, the total average + concentration of ammonium in the reactor at time=1 was 5.12±0.12mM NH4 . Therefore, there was 0.32mM more of ammonium, than the one needed for a balanced reaction of + anammox activity (4.8mM NH4 ).

Nitrate production during the first week began stable, with an average concentration of 2.04 ±0.21mM (day 1), and gradually started to decrease reaching a concentration of 0.96±0.22mM in day 3. In the second week nitrate was almost depleted from a concentration of 36µM at time=0 to 0µM after 7 hours.

Amino acids consumption

1a 1b 0.25 0.3 Serine 0.2 0.25 Glutamate 0.2 0.15 Glycine 0.15 0.1 0.1 0.05

0.05 Amino acids acids Amino (mM)

0 0 0 2 4 6 8 0 2 4 6 8 Time (hours) Time (hours) 2a 2b 0.3 0.35

0.25 0.3 Serine 0.2 0.25 Glutamate 0.2 0.15 Glycine 0.15 0.1 0.1 0.05

Amino acids acids Amino (mM) 0.05 0 0 0 2 4 6 8 0 2 4 6 8 Time (hours) Time (hours) 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).

In Figure 13 it can be observed that 90% of the amino acids were consumed in approximately 5 hours. Even though glutamate seems to be depleted faster there is no significant difference among them. In the same away during the second week of enrichment with amino acids the consumption shows similar behavior.

B. Second enrichment experiment (gly-glu) Before starting the experiment the reactor was stabilized to ammonium limitation. It took approximately one week to go back to the ammonium limiting conditions, having a concentration of 0.33mM of residual nitrite before starting the experiment.

0,4

0,35

0,3

(mM) 0,25

- Nitrite 2 0,2 Ammonium

0,15

or NO or

4 0,1

NH 0,05 0 0 1 2 3 4 5 6 7 Time (days) Figure 14. Nitrite and ammonium concentration before amino acids enrichment

Microbial growth monitoring The growth in the reactor system was monitored by a daily measurement of OD (600nm) and also with protein analysis. In Figure 15 a similar tendency can be observed with both measurements. 0,74 0,35 OD600 0,72 0,3 mg protein/mL 0,7 0,25 0,68 0,2

OD600 0,66 0,15

0,64 0,1 mgprotein/mL 0,62 0,05 0,6 0 3 5 7 9 11 13 day Figure 15. Microbial growth monitoring before amino acids enrichment.

7 0.4 6 0.35 5 0.3 day 1

4 0.25 (mM) (mM) day 2 3 0.2 0.15 day 3 2 0.1 day 4 1 0.05 0 0 0 2 4 6 8 0 2 4 6 8 Time (hours) Time (hours)

0.1 0.09 0.08 0.07 day 1

0.06 day 2 (mM) 0.05 day 3 0.04 day 4 0.03 0.02 0.01 0 0 2 4 6 8 10 Time (hours)

Figure 16. Nitrate, nitrite and ammonium concentration of one week experiment.

The experiment was monitored for 4 days in a row for only one week. In this experiment a similar behaviour was observed compared to the first experiment, starting with a mean value of residual nitrite of 0.31±0.02mM at day 1 and a decrease of 0.2mM during day 2 of the experiment. At day 4 the nitrite concentration remained at low concentrations of 0.1±0.01mM. Similarly to the first experiment, nitrite appears in high concentrations at day 1, and during the following days at time=0h there is residual nitrite, indicating that the reactor went back to ammonium limitation overnight and when the amino acids were added the residual nitrite was used. Ammonium started to accumulate gradually reaching a concentration of 0.07mM at day 4, whereas nitrate, did not appear to decrease in large amounts, until day 4 with a final concentration of 0.13mM. The microbial growth in the reactor in day 1 was of 0.319±0.085mg protein/mL and decreased to 0.114mg protein/mL. As a result of contamination with protozoa from the no sterilized solution of amino acids.

1a 1b 0.45 0.45 0.4 0.4 0.35 0.35 0.3 0.3 Glycine 0.25 0.25 Glutamate 0.2 0.2 0.15 0.15 0.1 0.1 0.05 0.05 Amino acids acids Amino (mM) 0 0 0 2 4 6 8 0 2 4 6 8 Time (hours) Time (hours)

Figure 17. Amino acids consumption of one week experiment in first day (1a), and third day (1b).

In this experiment the amino acids were not readily consumed at the beginning of the enrichment (Figure17. 1a). The consumption was observed on day 3 of the experiment, at time=6h 100% of amino acids were depleted (Figure17. 1b).

C. Third enrichment experiment (gly-glu) The third experiment consisted of the enrichment with glycine and glutamate. The reactor was restarted with fresh biomass with a concentration of 0.436mg protein/mL (Figure18.).

1,2 0,7 OD600

1 0,6

0,5 mg protein/mL

0,8

0,4 0,6

0,3 OD600 0,4

0,2 mgprotein/mL 0,2 0,1 0 0 0 2 4 6 8 10 12 day

Figure 18. Growth monitoring before amino acids enrichment.

2a 1a 0.5 0.5 0.45 0.45 0.4 0.4 day 4 0.35 0.35

(mM) 0.3 day 1 0.3 day 13 0.25 day 2 0.25 day 14 0.2 0.2 0.15 0.15 0.1 0.1 0.05 0.05 0 0 0 2 4 6 8 0 2 4 6 8 Time (hours) Time (hours) 1b 2b 0.5 4 0.45 3.5 0.4 day 1 0.35 3 (mM) 0.3 day 2 2.5 day 13 0.25 day 4 2 day 14 0.2 1.5 0.15 0.1 1 0.05 0.5 0 0 0 2 4 6 8 0 2 4 6 8 Time (hours) Time (hours) 1c 2c 7 7

6 6

5 5

4 day 1 4 day 13 (mM) day 14 3 day 2 3 2 day 4 2 1 1 0 0 1 2 3 4 5 6 7 8 9 0 0 2 4 6 8 Time (hours) Time (hours) Figure 19. 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).

During the first week the residual nitrite concentration started high as in the previous experiments starting with a mean value of 0.324±0.033mM at day1. Afterwards a significant decrease of nitrite was observed from day 2 onwards, reaching a mean value of 0.0976±0.005mM during day14.

During the first week ammonium was all depleted, remaining in a concentration less than 0.03mM during the first week of the experiment. In the second week ammonium started to accumulate as in the previous experiments, with an average concentration of 2.4±0.45mM in day 14.

Regarding nitrate, it stayed at high concentrations, with a little decrease during the first week after day 1 of enrichment. However nitrate did not stopped being produced, during all the period of the experiment. From the second week it was observed a sudden increase, which was not continuous but it gave a sign of no complete consumption of it (Figure19.2c).

1a 1b 0.7 0.7 0.6 0.6

0.5 0.5 Glutamate 0.4 0.4 Glycine 0.3 0.3 0.2 0.2

0.1 0.1 Aminio acids acids Aminio (mM) 0 0 0 2 4 6 8 0 2 4 6 8 Time (hours) Time (hours) Figure 20. Amino acids consumption for two days enrichment day 2 (1a) and day 4 (1b).

The amino acids were not consumed from day 1 but from day 4 onwards, this was observed by an accumulation of amino acids with a concentration of 0.5825 and 0.55mM of glutamate and glycine respectively (Figure 20.1a). However the amino acid assimilation was slower compared to the previous experiments, reaching 50% of the amino acids consumed at t=8h on day4.

The microbial growth during the experiment showed a fluctuating behavior, however from day 1 to day 12 there was an increase of 0.13mg protein/mL.

Enrichment with N-labeled amino acids in the reactor

1a 1b 28N2 20 2 25 7 30N2 18 1.8 6 29N2 16 1.6 20 14 1.4 5

12 1.2 15 4

(µM)

(µM) (µM)

10 1 (µM)

2 2

2 2 2 2

2 2 3 N

10 N N

8 0.8 N

29 28 6 0.6 29 2 4 0.4 5 1 2 0.2 0 0 0 0 0 2 4 6 8 0 2 4 6 8 Time (hours) Time (hours) Figure 21. 15N-glycine enrichment in day2 (1a), and day 4 one week experiment (1b)

During the experiment of enrichment with glycine and glutamate, 15N-glycine was added at 29 day 2. The production of N2, was not significant with a concentration of 2.029 µM after 8 hours. However in the second feeding (day4) with 15N-glycine there was an increase in the 29 production of N2 with a concentration of 5.94µM in time=7, indicating anammox activity 14 30 using the amino group ( N) of the amino acid. As expected, there was no N2 produced.

28 N2 2a 2b 16 2.5 18 10 29 N2 14 16 9 2 8 12 14 7 12 10 1.5 6

(µM)

(µM) (µM)

8 5 (µM)

2 2

2 2 2 8 2

N N

6 4 N

28 29 6 29 3 4 0.5 4 2 2 2 1 0 0 0 0 0 2 4 6 8 0 2 4 6 8 Time (hours) Time (hours)

Figure 22. 15N-glutamic acid enrichment in day 2(2a) and day 4 of experiment (2b)

During the second enrichment with glycine and glutamate, 15N-glutamic acid was added. In 29 this experiment the production of N2 had a significant increase in day 4 with a concentration of 8.75µM at time=7. Compared to day2 where a small increase was observed with a concentration of 2µM at time=7. In both experiments it was confirmed the use of the amino group by anammox bacteria.

3.2 Activity Tests with labeled amino acids Two batch experiments were done to know the possible role of N-labeled amino acids with nitrate. In both experiments only one amino acid was added, the first experiment with 15N- labeled glutamate (Figure 23.) and the second experiment 15N-labeled glycine (Figure 24).

1 2 1000 40 1600 35 28 900 35 1400 30 800 30 29 700 1200 25

(µM) 25 1000 600 (µM)

(µM)

2 2 (µM)

500 20 2 800

2 2 N 15 2

28 400 28

15 N 600 N

300 29 10 400 10 29 200 5 100 5 200 0 0 0 0 0 2 4 6 8 0 2 4 6 8 Time (hours) Time (hours)

Figure 23. Nitrate and 15N-glutamate activity test (1), nitrate, 15N-glutamate activity test and nitrite (added at time=2) (2).

15 29 In the experiment with N-glutamate, an increase of N2 in a similar concentration in both experiments was observed, showing that nitrate might be reduced to nitrite making it available for the anammox process. On the other hand the addition of nitrite did not seem to enhance 29 the production of N2, which might be also, because deamination is the rate limiting step. 28 Regarding the production of N2, it might come from denitrification or DNRA of the unlabeled nitrate followed by dinitrogen gas formation which might not be likely to have occurred. The consumption of 15N-glutamate was of 3.8 µmol ·mgprotein-1 h-1 without the addition of nitrite and 2.14 µmol ·mgprotein-1 h-1 with the addition of nitrite, this confirms that deamination is the limiting step.

1 50 1.8 2 300 8 1.6 7 28 40 1.4 250 6 1.2 29 30 200

1 5

(µM)

(µM) (µM)

150 4 (µM)

2 2 2 2 0.8 2

20 2

N N

0.6 3 N

28 29

28 100 29 10 0.4 2 50 0.2 1 0 0 0 0 0 2 4 6 8 0 2 4 6 8 Time (hours) Time (hours)

Figure 24. Nitrate and 15N-glycine activity test (1), nitrate, 15N-glycine activity test and nitrite (added at time=2) (2).

In the experiment with 15N-glycine, a pronounced increase was seen with the addition of unlabeled nitrite after two hours of incubation (Figure 24.2). However such results cannot be related to the activity since the amino acids were almost not consumed.

3.3 Fluorescence in situ hybridization (FISH) analyses The probability of a co-culture in the reactor was a possibility that wanted to be elucidated by phylogenetic analyses. At first place it was performed FISH analysis to target anammox bacteria AMX820 (cy3, orange) and all bacteria EUBmix (FLUOS, green) in the first enrichment. A B

C D

Figure 25. 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

During the enrichment with glutamate, serine and glycine, the presence of anammox coupled with other bacteria was observed the. However anammox bacteria remained predominant in the reactor population. Also a similar type of bacteria was observed together with anammox bacteria in the different samples taken.

The following figure21 shows the change in population at different stages during the enrichment of 2 weeks of glutamate and glycine. The probes used to target anammox bacteria AMX820 (FLUOS, green) and all bacteria EUBmix (cy3, orange).

E F

G HH

Figure 26. 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

During the enrichment with glycine and glutamate, the reactor population was monitored from the time of no addition of amino acids and the change after 14 days of enrichment.

What could be observed is the gradual increase of the other bacteria present in the reactor after 14 days of enrichment. However anammox bacteria remained predominant in the overall population in approximately 70-80%.

As a result of the clone library construction we found that the bacteria present in the reactor belong to the class β-proteobacteria. Therefore to confirm their presence in the overall period of enrichment; FISH analysis were done targeting anammox bacteria AMX820Cy5 (blue), and β-proteobacteria BETA420Cy3 (red). For this analysis, different days of enrichment were chosen, to get an indication whether the same bacteria were growing in every experiment. What can be observed is the presence of the same rod shape β-proteobacteria in the different samples, indicating a possible coupling activity of anammox bacteria with a β-proteobacterium.

I J

K L

Figure 27. 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.

Phylogenetic tree for 16S A clone library was construced to get an idea about the phylogentic relationships of bacetria inside the reactor system. The seven sequences obtained were all affiliated to the phylum Proteobacteria, except for the 16S.3 sequence which was found to be affiliated to the phylum Firmicutes. Thus the tree library was constructed according to the family of the most closely related microorganism found in the sequences: Azospira restricta. Such bacteria have the following classification: Phylum: Proteobacteria Class: β-proteobacteria Family: Rhodocyclaceae

Figure 28. Maximum likelihood phylogenetic tree of Betaproteobacteria (Rhodocyclaceae) 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. Clostridium sp. was used as an outgroup.

Phylogenetic tree for NirS The construction of the tree involved the hits obtained in the blast and other nitrite reductase bacteria that possess the gene encoding for NirS. Three of the seven sequences were found to be affiliated with a similar nitrite reductase uncultured bacterium, while other three sequences were affiliated to diverse nitrite reductase uncultured bacterium. What was observed is no relation with the 16S results, since no close affiliation was found with Azospira restricta.

Figure 29. 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.

Phylogenetic tree for NirK The construction of the tree involved only three NirK protein sequences. The blast results showed no similarity with Azospira restricta. Nevertheless is not shown a close affiliation with another microorganism. Thus the possible presence of this enzyme could not be dominant.

Figure 30. 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.

Chapter 4. Discussion The anammox metabolism has been an important research topic that has encountered several unknown scenarios. After their discovery the knowledge about the anammox process was far from complete and over time the deep understanding of the processes they could develop started to be elucidated, through activity assays and phylogenetic analyses. Until recently the different alternative pathways were studied, but still some research questions came up in the field. For this reason one of the research questions that came up while studying anammox metabolism is the occurrence and survival of anammox in the marine ecosystem, giving a step forward for this study.

Anammox bacteria activity The experiments done in this study showed the possible mechanism that anammox might perform in the presence of an alternative source of ammonium (amino acids). According to the results, there is a tendency in the response to the amino acids feeding. Where nitrite and nitrate concentration decreases, and ammonium accumulates over time. The possible mechanisms that might be occurring will be discussed for every nitrogen compound: 1. Nitrite: the sign of ammonium limitation is the presence of residual nitrite therefore the decrease of nitrite concentration indicates the possible ammonium for anammox process or that the consumption of nitrite by another microorganism. However there was always more ammonium present than the stoichiometric with a ratio of 1.24±0.5, instead of 1. This can led to a nitrite limitation scenario, where the ammonium becomes more available and the lack of nitrite limits anammox process. 2. Nitrate: anammox growth is related to the production of nitrate in every reactor system (Kartal et al., 2013). What was observed in the experiments was a decrease in nitrate concentration. There are two possible mechanisms occurring, one is the consumption of nitrate by another microorganism, the other possibility is that there is no net production or a lower production of nitrate by anammox bacteria. The latter mechanism cannot be completely stated because such scenario was not observed in all the experiments. In the last experiment nitrate concentration was always present, but in lower concentrations than in a standard reactor system. A reason might be that anammox population was relatively higher than in the previous experiments.

3. Ammonium: ammonium was completely consumed in the beginning of each experiment since that was a desired condition: anammox bacteria should be starved in ammonium. At the moment of adding amino acids it can be assumed that the amino group of the amino acids is released and becomes available to anammox bacteria. This is the case in the beginning of the experiments, but in time ammonium starts to accumulate possibly because of a complete release of the amino group of the amino acid, and a possible lack of nitrite for the production of dinitrogen gas. On the other hand, considering that anammox can also perform DNRA (Kartal., 2008)., ammonium accumulation can also be a result of such mechanism

The activity rate, in each experiment varied because of biomass concentration. However this only affected the time of activity, for the last experiment it took more time to observe the amino acid consumption. On the other hand it is important to consider the side population might take some time for acclimation and start cooperating with anammox.

In the stable isotope experiments the use of the amino group in the production of dinitrogen gas by the anammox process was confirmed. Moreover it was observed that the activity was limited by the deamination process, which indicates a lag-phase period. Consequently the consumption was seen after 1 or 2 days depending on the biomass concentration. The amino acid assimilation was determined in every experiment as complete over time, where it was assumed that the amino group of the amino acid was released and readily consumed by anammox or somebody else. In the catabolism of the amino acids, different products can be obtained, recalling what was found in the Kuenenia genome. The products formed such as pyruvate and 2-oxoglutarate, have an important role as intermediates in the citric acid cycle. Thus a further study that can be done is the determination of the uptake of the carbon group of the amino acids by anammox bacteria. Even though anammox bacteria are known as chemolithoautotrophic bacteria, it has also been addressed the versatile lifestyle that they can adopt under certain conditions (Kartal., 2008). Therefore 13C-labeling experiments could be used to determine the incorporation of the labeled carbon into the cells of anammox bacteria, through a lipids analysis of anammox specific biomarkers.

Microbial population During the experiments change in the colour of the reactor of light orange to turbid orange was observed and the depletion of nitrate which is not common in standard anammox activity. This gave an indication of the presence side population in the reactor system. As is already known, anammox bacteria cannot be grown 100% pure, because of being slow growing bacteria. Therefore in a reactor system there is the opportunity of a coupling mechanism, which was identified during the experiments. According to the phylogenetic analysis, in the results for the 16S sequences, a close relationship to the bacteria Azospira restricta was identified. Azospira restricta is a nitrogen fixing Betaproteobacterium, which was first isolated from groundwater samples (Bae et al., 2007). According to the description investigated they are heterotrophic but their growth is not favoured by the presence of amino acids, which contradicts the results of their growth in amino acids enrichment. In their characterization Azospira restricta were differentiated from Azospira oryzae by their inability to use nitrate as terminal electron acceptor and the inhibition of growth in the presence of organic acids. Therefore the studied features of Azospira restricta do not completely match with the activity and the environmental conditions in which this study was developed. This suggests the possibility of a not precise characterization of the bacteria or the need of a more thorough study to confirm their presence. Thus, further studies should be performed, the transcriptome analysis can give a more detailed information of the microorganism present and there possible metabolic activity. Since the depletion of nitrate suggested the possible presence of a denitrifier, it was decided to construct a tree library to identify the occurrence of the genes nirS and nirK. Denitrifying bacteria can possess either nitrite reductase (NirS) or copper-nitrite reductase (NirK) enzymes. The phylogenetic analysis of nirS provides an overview of the presence of one of the enzymes in the hits found. It was expected to find a possible match with Azospira in either nirS or nirK tree which was not encountered. However a recent study found the presence of NirS in Azospira sp., in such study a different primer was used (Heylen et al., 2012). This could explain the no correlation of the microorganism found for the nirS sequences to Azospira..

Regarding nirK, the sequences were not completely representative to identify the presence of it, since the PCR amplification was poor, but still there could be a minor population.

In addition, it has also been studied that there can be no complete correlation of the nirS or nirK tree topology since it studies have also found the occurrence of horizontal gene transfer between different taxa (Heylen et al., 2012). In order to confirm that the found nirS sequences belong to the same organism as the 16S gene sequences further analysis should be done, like the use of other primers for nirS amplification.

FISH analysis offered a view on the population diversity in the reactor which was observed to remain stable during the different enrichments. The presence of anammox bacteria with the side population of Betaproteobacteria was identified at the same. In the experiment maintaining anammox bacteria as the dominant organisms was desired, which was partially attained as a pulse feeding system of addition of amino acids. But if added continuously in higher concentrations the population might become dominant for the side population. Then, if a lower solids retention time (SRT) is applied to 10 days we could observe whether the amino acids favour anammox for their growth (Russ., personal communication). Such studies could provide more information about anammox activity in amino acids enrichment.

Perspective of application Since the beginning of anammox discovery its application has been of great interest for nitrogen removal in wastewater treatment, however some difficulties were encountered when applying anammox. Presently there have been approached new techniques for improving the performance of anammox for industrial application, like reducing the start up operation of the biological reactor through SHARON (single reactor system for high-rate ammonium removal over nitrite) process (Kuenen., 2008).

The determination of Total Nitrogen in the effluent in wastewater treatment is of great importance concerning its fate when disposed. In addition organic nitrogen has been found between 24-45% of the Total Nitrogen in the primary effluent where DFAA can also be present (Czerwionka et al.,2012). Further investigations showed evidence of free aminoacids in wastewater influent that might not be removed efficiently and can result in a problem if available for algae, generating the undesired eutrophication (Pehlivanoglu & Sedlak., 2004). It has been found that DFAA are available directly for algae uptake, which may come from the

42 effluent in wastewater treatment, the content of such compounds comprehends 10-20% of DFAA and Dissolved combined amino acids. (Pehlivanoglu-Mantas & Sedlak., 2008).

Furthermore several studies have put interest in the co-existence of anammox and denitrification for the simultaneous nitrogen and carbon removal. Several issues have been encountered in such mechanism, particularly in the nitrite competition. However in environmenal conditions the coupling activity of anammox and denitrification has been confirmed (Kumar&Lin, 2010). Thus the attainment of a stable co-culture system adapted to a wastewater treatment plant can provide advantages for their application.

Chapter 5. Conclusion The anammox process has been an interesting topic from the microbiological, ecological and industrial perspective. Therefore effort has been put in the deeper and broader understanding of the anammox process. This study was focused on the ecological area, more precisely in the marine environment where anammox bacteria have been identified to be important contributors for the removal of fixed nitrogen, under ammonium limitation environment. The presence of DFAA in the marine ecosystem has been found in some studies, and thus it was suggested that could be an alternative source of ammonium for anammox activity.

Therefore, through this study wanted to be elucidated the interaction that anammox bacteria might adopt under ammonium limiting conditions in the presence of amino acids. What was found in the research is that anammox uses the amino group that is released from the amino acid. However the cleavage mechanism that might be occurring was not clear because of the appearance of a side population. On the other hand it has been reported the interactions that anammox bacteria can adopt with other microorganisms such as ammonia-oxidizing crenarchaea in the Black Sea. Consequently the perspective of a coupling activity is an important approach because of a non-pure culture state of anammox bacteria. In order to unfold the question whether anammox bacteria can make use of organic carbon, further investigations can be performed. Through 13 C-labeled experiments and activity monitoring, where an alternative design can be developed adding continuously amino acids but in lower amount than the necessary to compensate the ammonium limitation.

From the industrial point of view the use of anammox bacteria in wastewater treatment plants has had an important impact for nitrogen removal. As a result the deeper understanding of anammox metabolism can provide broader knowledge for the design when applied.

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Annexes

Annex 1

Fixation for fluorescence in situ hybridization (FISH) (Retrieved from wiki.science.ru.nl,2014) Paraformaldehyde fixative

 Heat 66 ml milliQ water () to 60 degrees C in a water bath ().  Add 4 g of paraformaldehyde, the mixture becomes milky.  Gently add 2M NaOH until the mixture clears up.  Fill up to 100 ml with 3 x Phosphate Buffered Saline pH 7.4.  Cool down and set pH to 7.0-7.4 with 1M HCl  Filter the solution through a syringe filter

Procedure

 Mix the sample with 900 μl paraformaldehyde fixative (may be found in fish freezer top shelf).  Incubate on ice for 1 to 3 hours.  Centrifuge 3 minutes at maximum speed and remove the supernatant.  Wash 2 times with 1x PBS.  Resuspend in 100 % ethanol and 1x PBS (1:1, usually 500 μl each).  Store at -20 °C

Hybridization procedure

Sample Preparation

 Number slides with pencil.  Put 10 µl paraformaldehyde fixed sample onto the slides.  Air/N2 dry paraformaldehyde fixed sample (1-10 µl) on slide.  In the mean time, prepare the hybridization buffer and keep at room temperature, see below.  (Agarose: Put 5 µl agarose (0.1%) on top of the sample)

 Dehydrate sample on the slide in an increasing ethanol series (3 minutes each in 50, 80 and 100 % ethanol) and then dry slides standing up.  Possibility to store slides at -20°C.

Hybridization

Hybridization buffer (While dehydrating in ethanol) Pipet into an Eppendorf tube:

 180 µl 5M NaCl  20 µl 1M Tris/HCl pH 8.0  add formamide and MQ, depending on the applied stringency: % Formamide (v/v) Formamide (µl) MQ (µl) 0 0 800 5 50 750 10 100 700 15 150 650 20 200 600 25 250 550 30 300 500 35 350 450 40 400 400 45 450 350 50 500 300 55 550 250 60 600 200 65 650 150

 2 µl 10% (w/v) SDS  Prepare hybridization chambers by folding a piece of unbleached toilet paper and putting it into a 50 ml tube.  Put 10 µl hybridization buffer onto wells.  Add 1 µl of each probe (working solution 5 pmol/µl for Cy3 and Cy5 labeled probes or 8.3 pmol/µl for FLUOS labeled probes) and carefully stir with pipette tip.  Pour the remaining hybridization buffer into the hybridization chamber, immediately transfer the slide to the hybridization chamber and incubate 1.5 hours in the 46°C oven.  In the mean time, prepare the washing buffer and preheat this in the 48°C water bath.

Washing

Washing buffer Mix in a 50 ml tube:

 1 ml 1M Tris/HCl pH 8.0  5M NaCl and 0.5M EDTA pH 8.0 corresponding to the following table: (% Formamide) (NaCl (mol/l)) NaCl (µl) EDTA (µl) 0 0.900 9000 0 5 0.636 6300 0 10 0.450 4500 0 15 0.318 3180 0 20 0.225 2150 500 25 0.159 1490 500 30 0.112 1020 500 35 0.080 700 500 40 0.056 460 500 45 0.040 300 500 50 0.028 180 500 55 0.020 100 500 60 0.008 40 500 70 0.000 0 350

 Fill to 50 ml with MQ.  Rinse the hybridization buffer with some of the washing buffer from the slide. Incubate the slide in the remaining washing buffer for 20 minutes in the 48°C waterbath.  Remove the washing buffer with precooled distilled water and dry the slide with compressed air or briefly in the 46°C oven.

Microscopy / Storing

 Possibillity to store slides at -20°C.  Put 3 drops of vectashield onto the slide (not direct onto the wells) and put a coverslip on top.  Look at cells with the Zeiss microscope.

Annex 2 High Pressure Liquid Chromatography system (HPLC) Separation of amino acids (Retrieved from science.ru.nl, 2014) In the Varian 920-LC amino acids analysis involves derivatization with FMOC-Cl in a precolumn and separation with a reversed phase column and a High Pressure Liquid Chromatography system (HPLC). The detection limit for most amino-acids is about 10 pmol. In this case the sample should be delivered dried in a conical insert for a 2 ml HPLC-vial. In case of higher amounts, for example 150 nmol/amino acid, the sample should best be delivered dried in a closed flask, tube or HPLC-vial. The risk for contamination is much smaller with high-amounts samples.

 The following "protein- amino acids" can be determined cysteine acid, arginine, hydroxyproline, serine, asparagine acid, glutamine acid, threonine, glycine, alanine, proline, methionine, valine, phenylalanine, isoleucine, leucine, cysteine,hydroxylysine, histidine, lysine and tyrosine.  Also "physiologic amino acids" can be analyzed: 1-methylhistidine, 3-methylhistidine, anserine, asparagine, glutamine, citrulline, taurine, amino-adipinezuur, ethanolamine, beta-alanine, gamma-aminobutyric acid, alpha-aminobutyric acid, beta-amino-isobutyric acid, tryptophane, carnosine and ornithine.  Norleucine or norvaline can be used as internal standard, e.g. for quantification (these are prevailed because of their better separation compared to other amino acids). Components The amino acid analysator consists of the following components:  ProStar 420 autosampler (meant for the pre-derivatisation with Fmoc-Cl ).  The Varian 920-LC solvent delivery system, with a dual wavelength UV-VIS detector, a fluorescence detector, column oven and a low pressure quaternary gradient pump.  Conditions of analysis: Type of column: Varian Pursuit XRs Ultra C18 Stationary Phase : Pursuit XRs Ultra C18 Column temp: 36oC

UV detection: 269 nm Fluorescence detection: 265/340 nm Acquisition: 35 min Flow: 0.5-0.6 ml/min

Annex 3

PCR amplification The following figure shows the results obtained in the amplification fragments and the marker used.

The author of this thesis was born in Mexico City on the 11th of August 1988. In 2010 she received the Bachelor degree of Food Engineering from Universidad Autónoma Metropolitana (UAM-I). Afterwards she collaborated in several projects in the research group of Dr. Sergio Revah in the Environmental Biotechnology department at the same university. This experience enhanced her motivation and enthusiasm in the environmental biotechnology field. In 2012 she got accepted to study an Erasmus Mundus programme International Master in Environmental Science and Technology (IMETE), with a partial scholarship from the mexican government. During the 2 year program she was formed academically in Delft, Prague and Ghent. Afterwards she concluded with her master thesis at Radboud University, in the research group of Mike Jetten. There she got involved in the understanding and study of anammox metabolism. Such project reinforced her motivation in the multidisciplinary field and love for science.