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Dissertation DISSERTATION Titel der Dissertation The ecophysiology of nitrite-oxidizing bacteria in the genus Nitrospira: Novel aspects and unique features angestrebter akademischer Grad Doktor der Naturwissenschaften (Dr. rer. nat.) Verfasser: Frank Michael Maixner Matrikel-Nummer: 0409617 Dissertationsgebiet (lt. 444 Ökologie Studienblatt): Betreuer: Univ.-Prof. Dr. Michael Wagner Wien, am 13. April 2009 meiner Familie Contents Chapter I General Introduction and Outline 1 Chapter II Nitrite concentration influences the population structure of Nitrospira-like bacteria 27 Chapter III Environmental genomics reveals a functional chlorite dismutase in the nitrite-oxidizing bacterium ‘Candidatus Nitrospira defluvii’ 39 Chapter IV Nitrite oxidoreductase (Nxr) - the metabolic key enzyme of nitrite-oxidizing Nitrospira: Characterization of an unusual Nxr and its application as a novel functional and phylogenetic marker gene 67 Chapter V Summary/Zusammenfassung 123 Appendix 131 Supplement chapter I – paper 1 Selective enrichment and molecular characterization of a previously uncultured Nitrospira-like bacterium from activated sludge 133 Supplement chapter I – paper 2 Physiological and phylogenetic characterization of a novel lithoautotrophic nitrite-oxidizing bacterium, ‘Candidatus Nitrospira bockiana’ 145 Supplement chapter I – figure 1 155 Supplement chapter I – table 1 161 List of publications 163 List of oral & poster presentations 165 Danksagung 169 Curriculum vitae 171 Abbreviations A adenine AmoA ammonia monooxygenase subunit A ARB Arbor (software package comprising various tools for sequence and phylogenetic analysis) AOA ammonia-oxidizing archaea AOB ammonia-oxidizing bacteria C cytosin CLD chlorite dismutase CLSM confocal laser scanning microscope; confocal laser scanning miocroscopy DNA deoxyribonucleic acid et al. et alii Fig. figure FISH fluorescence in situ hybridization G guanine Hao hydroxylamine oxidoreductase N nitrogen N. defluvii “Candidatus Nitrospira defluvii” NOB nitrite-oxidizing bacteria Nxr nitrite oxdioreductase PCR polymerase chain reaction RNA ribonucleic acid rRNA ribosomal ribonucleic acid T thymine t ton U uracile wwtp wastewater treatment plant Chapter I General Introduction and Outline Chapter I Front: 3D visualization of the nitrifying community in a biofilm sample stained by fluorescence in situ hybridization with the probes S-*-Ntspa-1431-a-A-18 (sublineage I Nitrospira, labelled with Cy3, red), S-*- Ntspa-1151-a-A-20 (sublineage II Nitrospira, labelled with FLOUS, green) and NSO1225 (ammonia-oxidizing bacteria, AOB, labelled with Cy5, blue). AOB and nitrite-oxidizing bacteria (NOB) of the genus Nitrospira are in close vicinity to each other. Note the different spatial arrangement of the two Nitrospira sublineages relative to AOB in the biofilm (see also Chapter II). 2 Chapter I General Introduction 1. The Global Nitrogen Cycle 100 years after Fritz Haber patented the process to “synthesize ammonia from its elements”, we nowadays live in a world which is highly dependent upon, and transformed by man-made fixed nitrogen (Erisman et al., 2008). At first the “Haber-Bosch” nitrogen allowed large scale production of explosives resulting in millions of war victims. Second the ammonia is a starting product in several sectors in chemical industries producing plastics and synthetics. Most of the fixed nitrogen, however, is used to produce fertilizer in the forms of ammonium nitrate, calcium nitrate, ammonium bicarbonate, and several mixtures of nitrogen and phosphorous containing compounds. Since 1960, accompanied by an increase of the world population by 78% (since 1970), large scale production of synthetic nitrogen fertilizer supports at least half of the human population by increased food supply (Centre for Ecology and Hydrology, 2008, Galloway et al., 2008) (Fig. 1). 1,4 × 108 7 × 109 World N-production 1,2 × 108 USA N-production USA N-imports 6 × 109 Other 8 1 × 10 Explosives Plastics and synthetics Fertilizers 8 × 107 5 × 109 World population 6 × 107 4 × 109 World population 4 × 107 Nitrogen content (fixed) [t] 2 × 107 3 × 109 0 2 × 109 1940 1950 1960 1970 1980 1990 2000 2010 Year Fig. 1: Trends in worldwide industrial ammonia production (via the Haber-Bosch process) and human population throughout the last 70 years. Partitioning of synthetic fixed N to different branches of production in the United States of America between the years 1975 and 2003 indicated in bars, color-coded according to the legend. [Data sources: http://minerals.usgs.gov/ (industrial N-production); Chamie, 1999 (world population)]. 3 Chapter I Beside the nitrogen emitted to the atmosphere during fossil fuel burning the two major sources of anthropogenic nitrogen released into the environment are fertilizers and wastewater. Less than 30% of the synthetic nitrogen in fertilizers reaches the end- products (Smil, 2001). The remaining nitrogen is either lost via emission of elemental nitrogen, nitric oxides and ammonia or through leaching of nitrate. To counteract this loss often more fertilizer is used in agriculture than needed. A recent study addressed the problem of excessive nitrogen fertilization in agricultural areas in China (Ju et al., 2009). Ju and coworkers tracked the fate of fertilizer nitrogen and could show that with a higher load of fertilizer, plants are less efficient at taking up synthetic nitrogen. As a result most of the nitrogen leached into the ground and surface water. A reduction of nitrogen fertilizer by two thirds and better fertilization timing would reduce environmental nitrogen contamination without compromising crop yields (Qiu, 2009). The extraordinary population growth in the twentieth century resulted in a dramatically increased nitrogen deposition into the environment. On the one hand there is the aforementioned excess of fertilizer load due to the increased demand for food. Furthermore, the increasing population causes a higher wastewater deposition and mainly in less developed regions this wastewater ends up untreated in the environment. Therefore, beside better nitrogen fertilizer management the efficient elimination of nitrogen in modern wastewater treatment plants is a crucial process to reduce the import of nitrogen compounds into the environment. The increased anthropogenic nitrogen deposition destabilized ecosystems in most parts of the world and resulted in major human health effects and unintended environmental consequences (Vitousek et al., 1997, Galloway et al., 2008). On the one hand, atmospheric reactions of emitted ammonia, nitric oxides, and sulfur oxides result in fine particle formation (Sharma, 2007), which increase the risk of cardiovascular and pulmonary diseases (Dominici et al., 2006, Neuberger et al., 2007). Additionally, nitrogen-compounds like ammonia and nitrite are highly toxic to aquatic life (Arthur et al., 1987) and elevated nitrite and nitrate levels in drinking water can have severe health consequences to humans (Schneider & Selenka, 1974, Ward et al., 2005). Beside these direct toxic effects of several nitrogen-compounds on living organisms, raised deposition of anthropogenic nitrogen compounds into natural habitats causes fatal ecological damages. Some environmental effects particularly noteworthy are coastal eutrophication due to import of significant amounts of nitrogen through rivers (Howarth & Marino, 2006) resulting in anoxia and hypoxia of the sea bottom (Rabalais, 2002, Rabalais et al., 2002) or elevated nitrogen deposition linked to eutrophication of different 4 Chapter I natural areas resulting in a considerable loss of floral biodiversity (Phoenix et al., 2006). Beside the eutrophication of terrestrial and aquatic systems the interaction of the nitrogen and carbon cycle and the resulting global acidification has already and will further have severe consequences on earth climate (Gruber & Galloway, 2008). Recently, for example, aquatic invertebrates in nitrate-rich environments have been shown to emit the potent greenhouse gas nitrous oxide in quantitatively important amounts (Stief et al., 2009). In summary, this manmade nitrogen deposition caused an ecological imbalance into many ecosystems and the effects would have been even more dramatic without the high buffering capacity of nitrogen cycling microorganisms. However, to effectively monitor ecological changes due to anthropogenic pressure a better understanding of the mechanisms behind the global nitrogen cycle is urgently needed. It is important to understand the nitrogen cycle at a range of scales of biological organizations, beginning with the vast microbial diversity involved in nitrogen conversion and their interactions (Horner-Devine & Martiny, 2008). Especially during the last decade, groundbreaking and surprising findings in microbial ecology such as bacteria capable of anaerobic ammonia oxidation (ANAMMOX) (Mulder et al., 1995) or the existence of ammonia oxidizing archaea (Treusch et al., 2005, Könneke et al., 2005) have shown that our knowledge of the nitrogen cycle and the involved microbial key players is still scarce. The following sections will focus on nitrite-oxidizing bacteria (NOB) of the genus Nitrospira, one of the aforementioned key players in the nitrogen cycle but yet less intensively studied groups of NOB. 5 Chapter I 2. The Biological Nitrogen Cycle: Microorganisms keep the wheel turning All forms of life require nitrogen as integral part of proteins and nucleic acids, and thus every living organism participates in the nitrogen-cycle. Microorganisms, however, catalyse essential steps
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