Ongoing global warming particularly effects Arctic ecosystems with increased glacial retreat and associated changes in run-off. However, how aquatic nitrous oxide (N2O) fluxes will change in High Arctic lakes and catchments remains uncertain. As the third most important greenhouse gas and major ozone-depleting substance in the stratosphere, N2O is a crucial parameter in order to understand feedback mechanisms on climate. We will identify N2O and NO3- sources as well as production and consumption processes in lake Revvatnet, streams, glacier run-off and ponds surrounding the Polish research station at Hornsund, Svalbard using state-of-the-art molecular techniques (qPCR) and NO3- and N2O isotopic composition. This project will provide the first estimate of water-to-air transfer of N2O in high Arctic lakes and catchments in Svalbard. Considering that climate change is expected to drastically alter the input of inorganic nitrogen (N) sources, this study represents an important contribution to the understanding of the biogeochemistry of the region and to establish a baseline to assess future change in nutrient regime for this climate sensitive region.

The great majority of global lakes is oversaturated with respect to carbon dioxide (CO 2 ), methane (CH 4 ) and nitrous oxide (N 2 O). These lakes are recognized as a globally significant source of greenhouse gases (GHGs) releasing annually up to 560 Tg CO 2 -C, 220 Tg CH 4 -C, and 0.6 Tg N 2 O-N to the atmosphere. Traditionally, the lacustrine GHGs oversaturation has been considered to result mostly from the metabolism of terrestrial organic matter (OM) imported to lakes from their catchments. However, some high-latitude and -altitude lakes may seasonally become undersaturated in CO 2 and N 2 O, thus possibly turning into a sink for the atmospheric GHGs, which has been largely understudied. In fact, we anticipate that GHG saturation levels in these remote lakes are likely to fluctuate strongly depending on the efficiency of cross- and within ecosystem connectivity effects that modulate the OM transport, as well as on the efficiency of in-lake transport, ventilation, and exchange between sediments and the water column. This project, thus, aims to improve our understanding of how climate-controlled connectivity effects across and within aquatic, terrestrial and glacial environments influence the organic matter flow and greenhouse gas (GHG) production and emission in high-latitude and -altitude lakes, as well as to incorporate resulting estimates for these environments in global carbon and GHGs budgets. Anticipating that the climate in the Arctic and Alpine regions is changing two to three times faster than anywhere else on our planet, we will develop and calibrate a process-based lake metabolism model, combining paleolimnological data, real-time limnological monitoring and field investigations, as well as experimental simulations of extreme meteorological and hydrological events, to more accurately predict future feedback effects between organic-matter processing rates and fluxes and the GHGs saturation levels across sediment-water and water-atmosphere interfaces in high latitude and altitude regions. The particular goals of this project will be reached by addressing 15 research hypotheses, centered around interactive mechanisms responsible for climate- and connectivity-dependent changes in the regimes of organic matter import and nutrient availability in lakes, as well as anticipated links to changes in water transparency, stratification strength and phenology. The potential mechanisms that we plan to investigate include: 1) longer-term temperature effects that enhance organic matter import to Arctic and Alpine lakes, thus stimulating microbial respiration and GHG production in the water column and sediments; 2) shorter-term effects of higher rainfall rates and warming-enhanced cross-ecosystem connectivity that can lead to large pulsed OM inflows into lakes and, thus, can disproportionately stimulate CH 4 and N 2 O production rates; 3) extension of the ice-free period in lakes and catchments, through the effects of warming and higher rainfall, allowing for higher OM inflows and longer periods when CH 4 and N 2 O can be directly released to the atmosphere; 4) effects of atmospheric warming that, in turbid lakes, result in "thermal shielding" leading to the extension of anoxia in bottom waters and sediments, which is conducive to higher CH 4 and N 2 O production; 5) warming-enhanced cross-ecosystem "feeding" allowing for a more labile fraction of OM to be imported to lake waters and sediments where it can fuel CH 4 and N 2 O production; 6) more efficient transport of nutrients across ecosystem boundaries that allows for a higher phytoplankton production, thus stimulating biological CO 2 uptake and creating more substrate for methanogenesis; 7) greater CO 2 depletion in the Arctic and Alpine lakes through weathering-associated chemical uptake. Overall, we anticipate that, as a consequence of the ongoing environmental changes and altered cross- and within-ecosystem connectivity, higher summertime primary production at the surface and greater OM burial in the sediments will ultimately lead to enhanced production and emissions rates of potent GHGs, such as CH 4 and N 2 O, from Arctic and Alpine lakes. Considering that CH 4 is about 30- and N 2 O about 300-times more potent then CO 2 (in terms of global warming potential), enhancement of ecosystem connectivity, likely leading to the increasing release of these potent GHGs from remote lakes, will have implications for their future contribution to the global GHG budget and the atmospheric greenhouse effect.

Bioavailable nitrogen (N) controls marine biological productivity and thus the capacity of the global ocean to sequester atmospheric CO 2 in the abyss through the production and remineralization of sinking algal organic matter. In lakes, high concentrations of bioavailable N cause eutrophication, increased algal growth, and in turn oxygen loss. Past changes in the input/output and internal cycling of fixed N (e.g., nitrate) in the marine and lacustrine environments can be reconstructed by analyzing the N isotopic composition (the 15 N/ 14 N ratio, or d 15 N) of organic matter in the sedimentary record. Bulk sedimentary d 15 N signatures, however, can be biased by secondary alteration and external (e.g., terrestrial) N inputs, so that recently, the focus has shifted to measuring the d 15 N of organic N that is trapped and protected in the mineral structure of (micro-)fossils, such as diatoms, foraminifera and corals, which is thought to record the pristine N isotope signature of nitrate in the surface water. Yet, the validity of these new N isotope proxies is still under scrutiny, as the exact modulating controls during microfossil-bound N isotope signature generation remain uncertain. The overarching goal of the proposed study is to ground-truth the diatom-bound N isotope paleo-proxy in the marine and lacustrine environments through a combination of experimental and field studies. In a first work package, we want to investigate how the d 15 N signature of diatom frustule-bound N is acquired (i.e., how well it tracks the nitrate source) by determining the relationships among the d 15 N values of the nitrate source to the diatoms, the d 15 N of the bulk diatom biomass, and the d 15 N of diatom-bound N in laboratory diatom culture experiments, as well as in the modern ocean water column and in lakes . Thereby, we will also attempt to assess the effects of changing environmental conditions and diatom assemblages. In a second work package, focusing on lacustrine sediments, we will examine whether fractional decomposition in the water column and/or diagenetic (i.e., altering) effects in the sediment during early burial alters the pristine N content and the d 15 N signature of diatom-bound N over time. Towards this goal, we propose combined N isotope analyses of sediment trap, surface sediment, and downcore sediment material from a time-series of varved sediment cores from a lake in Sweden, as well as degradation experiments of diatom cultures . Finally, in a third work package, we want to explore, for the first time, the application of the diatom-bound d 15 N proxy in lacustrine sediments of Swiss lakes as a recorder of the eutrophication history over the past century. The proposed research will assess the integrity of diatom-bound N as a proxy for paleoenvironmental change in marine and lacustrine sediments. Furthermore, the combined analyses of bulk sediment d 15 N and diatom-bound d 15 N in the downcore records w ill shed light on the effects of early diagenesis on bulk sedimentary organic matter, and will allow us to reevaluate the use of bulk sediment d 15 N as a proxy for reconstructing the past N cycle in the oceans and lakes.

Nitrous oxide (N 2 O, also known as laughing gas) has become the third most important anthropogenic greenhouse gas, after CO 2 and methane. Oceanic N 2 O emissions to the atmosphere represent up to 35 % of the global natural sources. Freshwater N 2 O emission are less well constrained, due to high spatial and temporal variability of aquatic N 2 O fluxes from inland waters. The exact biogeochemical controls on N 2 O cycling are still poorly constrained. In order to understand changes in the magnitude of N 2 O fluxes from aquatic ecosystems in response to fluctuating biogeochemical conditions (i.e., redox state, dissolved nitrogen, and organic substrates), it is imperative to determine the individual contributions of the microbial (ammonium oxidation/nitrification, nitrifier-denitrification, and denitrification) and abiotic N 2 O production pathways and their sensitivity to changing environmental conditions. The potential niche overlap of denitrifiers and nitrifiers along oxygen gradients, or between ammonium oxidizing bacteria and archaea in freshwater, make it difficult to distinguish between the different N 2 O sources and their process-specific controls. For example, an increasing number of studies suggest that denitrification, mostly known as N 2 O sink in anoxic waters, is a largely overlooked N 2 O source in the suboxic water masses overlaying open ocean oxygen minimum zones. At the same time, despite the canonical view that N 2 O reduction is an anaerobic process, high abundances of N 2 O reduction genes and transcripts have been found in marine oxic waters indicating a potential unknown N 2 O sink in surface waters. Whether such an aerobic N 2 O sink exists also in lakes remained unaddressed. As for nitrification in surface/subsurface lake waters, the role of dissolved organic N compounds (i.e. urea and cyanate) as potential substrate and precursor in N 2 O production is uncertain. The aim is to identify and quantify specific N 2 O production and consumption pathways in lacustrine environments, and to provide insight into the key microbial players, pathways, dynamics and environmental controls on N 2 O cycling. We will shed light on the blurring redox boundaries and environmental controls (e.g., nutrient availability) that modulate net N 2 O production in a lacustrine environment, where autotrophic denitrification is the dominating N loss pathway (Lake Lugano, Switzerland). The main objectives of the proposed project are to: 1) Identify the seasonal and vertical variability of N 2 O consumption, on the relative importance of N 2 O production processes in the water column, the abundance of process marker genes/transcripts, and the N 2 O producing/consuming microbial community composition in the studied lake. 2) Assess the sensitivity of N 2 O production and reduction processes to changes in dissolved nitrogen substrate and oxygen availability. 3) Determine the importance of underappreciated organic N-sources such as urea and cyanate for biotic and abiotic N 2 O production in the lake water column. We will use 15 N/ 18 O-tracer incubation experiments for potential-rate estimates along with natural abundance isotope measurements of N 2 O, NO 3 - , NO 2 - , and NH 4 + , which will provide an integrated signal of overlapping processes that affect the stable isotope pools of these molecules over short and longer time periods. We will combine isotope-biogeochemical results with data on abundance and community composition of N 2 O producers and consumers. The results that we expect to come out of the proposed project will provide essential knowledge about the mechanistic regulation of different N 2 O production pathways in aquatic environments, and will thus help to improve and validate existing and future global models predicting lacustrine N 2 O emissions.

In many ocean regions, as a limiting nutrient, bioavailable nitrogen (N) controls marine primary productivity and thus the ocean's capacity to fix and sequester atmospheric CO 2 in its interior. In many lakes, N from both natural and anthropogenic sources is an important driver of eutrophication. Therefore, both in the ocean and in lakes, it is crucial to understand the sources and sinks of fixed N. Denitrification, the microbial reduction of nitrate to dinitrogen (N 2 ), and other modes of suboxic N 2 production (e.g., the anaerobic oxidation of ammonium, or anammox), are the most important sinks of fixed N in aquatic environments, but particularly with regards to the N cycle in the ocean, there is a persistent debate regarding the overall size of sinks and sources. Isotope ratios of nitrogenous species (e.g., 15 N/ 14 N) can provide important constraints on the natural N cycle. In order to use stable isotope measurements as a means to trace fluxes of N in aquatic systems, however, it is imperative to understand the isotope effects associated with these fluxes. While denitrification at the organism-level is known to be associated with a marked N isotope fractionation, the expression of the N isotope effect of benthic (i.e., sedimentary) denitrification in the water column above is only poorly constrained, and likely varies with the environmental conditions. Moreover, the possible impacts of other benthic N cycling processes on the N isotope exchange between the sediments and the water column (e.g., anammox, nitrate reduction to ammonium (DNRA), nitrate uptake, and/or nitrate regeneration) remain uncertain. Understanding the overall N isotope effect of net benthic N loss is a prerequisite for using N isotope measurements to infer its relative importance in the N cycle, in the global ocean or in a specific environment. Here we propose an in-depth investigation of the isotope effects of benthic fixed N elimination and nitrate regeneration in aquatic sediments. The prime goal of the proposed research is to build a thorough understanding of the modulating controls on the nitrate and nitrite N (and O) isotope signatures of denitrification and anammox (and the interacting effects from other benthic N cycling reactions), and the N isotopic composition of gaseous N (i.e., N 2 and N 2 O) that is ultimately lost from the sediments. We predict that the expression of the biological isotope effect of benthic N elimination at the level of sediment-water exchange will vary across different environments, and will strongly depend on the reactivity of the sediments, the O 2 penetration, the physical boundary conditions (i.e., diffusive transport), and on the extent to which other processes than denitrification contribute to the overall N cycling (nitrification, anammox, DNRA). Combining 1.) laboratory experiments with natural and artificial sediments, 2.) field investigations into the sediment porewater (N and O) isotope dynamics of distinct lacustrine and marine denitrifying benthic environments, and 3.) mathematical modeling , and making use of innovative multi-isotope techniques (natural abundance isotope analysis of NO 3 - / NO 2 - , ammonium, dissolved organic N, and N 2 O, as well as 15 N tracer experiments), we attempt to gain complementary information on how the combined isotope effects of benthic nitrate reduction and nitrate regeneration are expressed in the water column of lakes and the ocean. With the diagenetic porewater N isotope model that this project will deliver we will establish a quantitative framework for assessing benthic isotope fluxes and for verifying our hypotheses. The research proposed will result in the first comprehensive characterization of sediment pore-water N (and O) isotope dynamics in lacustrine settings, and will allow experimental constraints on the variability of N isotope effects during benthic nitrate reduction across different biogrochemical regimes. While the field component of the project focuses on lake sediments, the results expected will be directly pertinent to understanding of fixed-N elimination isotope effects in the ocean. It will thus be highly relevant for the use of N isotope measurements for local, regional, and even global N budgets, and will provide the basis for both paleolimnological and -oceanographic extrapolation.

Combining field-sampling and incubation-experimental efforts, and employing stable isotopic, radio-labelling, and molecular analyses applied to samples from Lake Lugano (Southern Switzerland), we specifically proposed to investigate CH 4 production related to the exploitation and decomposition of methylated organic compounds by establishing functional links between epilimnetic CH 4 accumulation, concentrations of methanogenetic substrates and nutrients, the molecular composition of dissolved organic matter (DOM), and the planktonic community structure in this eutrophic lake. During the tenure of the original project, we have broadened the scope of the original research plan by establishing a regular sampling of plankton and CH 4 biogeochemistry in another Swiss Lake, Lake Cadagno. During the first 2.5 years of the project, we generated a comprehensive data set that allowed us to answer important questions that we proposed in the parent proposal. Based on results from both lakes, we could demonstrate clear seasonal variability in the magnitude of the lacustrine methane paradox, and establish links between the accumulation of CH 4, feeding zooplankton, and the degradation of planktonic detritus. We could experimentally show that CH 4 can be produced in oxygenated lake waters from methylated compounds both under nutrient-limited and nutrient-replete conditions. At the same time, we were able to exclude the possibility that the lacustrine "methane paradox" in the two studied lakes results from an upwelling or lateral transport of sedimentary gas. Moreover, we were able to prove that aerobic CH 4 accumulation varied with different plankton communities, confirming close links to the lacustrine productivity cycle and general limnological conditions. For example, we observed putative links between the development of picocyanobacterial blooms ( Synechoccocus) , the concentration of dimethylsulfide (DMS, a potential substrate in CH 4 formation) in subsurface waters, and the accumulation of CH 4 , yet, the overall amount of DMS seemed insufficient to fully account for the observed methane paradox. While canonical methanogens are virtually absent from the epilimnetic microbial communities of both lakes (less than 0.05% of OTUs), methylotrophic bacteria as well as photoautotrophs are abundant, and are potentially capable of exploiting methylated nutrients (methylphosphonate - MPn, methylamines-MA). In fact, the most recent set of incubation experiments within SNSF project 3623910 supported that both methylphosponate (MPn) and methylamine (MA) are suitable substrates for CH 4 generation in oxic waters of the studied lakes. Most importantly, and in contrast to literature reports, the production of CH 4 was not inhibited by inorganic phosphorous (as PO 4 3 -P), indicating that P-limitation is not a pre-requisite for the exploitation of methylated P to occur in nature. Although the data so far are highly promising and provide a solid basis for at least three publications, not all of the questions originally posed could be addressed unambiguously (due to the highly complex nature of analytical techniques required to identify and quantify natural concentrations of methylated P and N compounds, as well as molecular characterization of DOM). Specifically, uptake of MPn and/or MA in the context of lacustrine aerobic CH 4 production, the identity of the microbes/algae involved, and the specific role of picocyanobacteria still needs further investigation. Given the comprehensiveness and great variety of data already generated, the project extension will provide the time required for a more in-depth statistical analysis and exploitation of the already existing data set. In addition, we propose complementary bio-geochemical analyses of samples that have already been collected during past samplings to confirm the robustness of existing results (characterization of DOM by FTIR-ICMS, characterization and quantification of polysaccharide esters of phosphonic acids by NMR and methylated amines by solid-phase microextraction techniques), as well as incubation experiments with 13 C labelled MPn and 15 N labelled MA to assess uptake and metabolism of these compounds by isolated Synechoccocus strains using isotope-ratio mass-sepctrometry and NanoSims imagining. Prime objective of the project extension will be to establish whether, and to what extend, the exploitation of dissolved organic phosphorus (DOP) and methylated-N compounds occurs naturally under different trophic conditions, and what the potential rates of CH 4 generation are. We will also investigate further the C isotopic fingerprint (including clumped CH 4 isotope signatures) of MPn or MA-based aerobic methane production. This project extension thus promises to provide additional milestones in our efforts to understand the "methane paradox" in lakes, helping us to gain insight into the biogeochemical controls on global CH 4 emissions from aquatic environments.

Molecular oxygen (O 2 ) is one of the most important electron acceptors for enzymatic and abiotic redox reactions in the environment. Enzymes that utilize O 2 as the terminal electron acceptor catalyze a large number of biological reactions involved in processes of metabolic activity, cellular detoxification, and biosynthesis. Many of these reactions are essential for life, but some of them also contribute to the metabolic and co-metabolic removal of anthropogenic organic contaminants in natural aquatic environments and during wastewater treatment. Abiotic reduction of O 2 is important for the biogeochemical cycling of elements, such as iron or other trace metals, as well as the photomineralization of organic compounds in sunlit surface water. Measuring systematic changes in the stable isotopic composition (i.e., isotope fractionation) of dissolved O 2 during enzymatic reactions has been shown to enable the identification of underlying O 2 reduction mechanisms. Consequently, measuring isotope fractionation of O 2 is a promising tool for investigating environmentally relevant redox reactions. However, to ultimately apply this approach to reactions with unknown O 2 reduction mechanisms, it is important to systematically extend the current knowledge of reaction-specific 18 O-kinetic isotope effects ( 18 O-KIEs), which ultimately determine observable isotope fractionation of enzymatic and abiotic reactions of O 2 in aquatic environments.The overall objective of this project is to experimentally determine reaction-specific 18 O-KIEs associated with selected enzymatic and abiotic O 2 reduction reactions and, in turn, to evaluate the potential of O 2 isotope analysis as a tool to study environmental redox reactions. The central part of this project will entail the systematic investigation of well-known enzymatic reactions in laboratory experiments to determine the dependence of O 2 consumption-associated 18 O-KIEs on the type of enzyme, substrate, and catalyzed reaction. Different O 2 reduction reactions will be studied in headspace-free reactors containing an oxidase or oxygenase enzyme, native or non-native substrates, and required cofactors and/or co-substrates. Reaction-specific 18 O-KIEs will be determined in replicate experiments, where O 2 concentrations will be monitored continuously, and the isotopic composition of dissolved O 2 will be measured at different time-points. Comparison of these 18 O-KIEs with isotope fractionation of O 2 during enzymatic oxidations of anthropogenic contaminants with unknown reaction mechanisms will represent the first step in our efforts to evaluate the suitability of this approach to study O 2 reduction mechanisms in anthropogenically impacted ecosystems. Similarly, 18 O-KIEs will be studied for photochemical reactions involving O 2 , including singlet oxygen and superoxide radical formation. The gas chromatography isotope ratio mass spectrometry (GC/IRMS) method used for these experiments will be optimized regarding sample size and the O 2 extraction procedure at the beginning of the proposed project.It is expected that results from this project will enable the routine use of isotope analysis of O 2 to study environmentally relevant redox reactions in laboratory-scale experiments and provide valuable mechanistic insights into specific enzymatic and abiotic redox reactions. The comprehensive set of 18 O-KIEs determined in this work will establish benchmarks for various abiotic and microbial O 2 consumption processes in the aquatic environment, which can be used in future studies applying isotope analysis of dissolved O 2 , or of organic products containing O-atoms derived from O 2 , in natural systems.

The Arctic Ocean is one of the prime sentinels of the Earth's climate. The (sub)polar Atlantic Ocean exerts important controls on the global ocean circulation, the distribution of nutrients in the ocean, and the meridional heat transport. Primary productivity (PP) is an important component of the ocean's biological carbon pump, controlling the partitioning of CO2 between the ocean and the atmosphere. The Greenland ice sheet (GIS) has been melting at unprecedented rates as a consequence of global warming, yet the potential impacts of ice melting on the nutrient availability and biological productivity of Greenland's marine ecosystem are still unclear, because physical and chemical effects are multifaceted, and can either operate in tandem or oppose each other. For example the release of meltwater at depth from marine-terminating glaciers can induce nutrient upwelling and fuel summertime phytoplankton blooms. In contrast, stratification induced by release of meltwater at the ocean surface can impede vertical nutrient supply, limiting primary productivity. Similarly, associated feedbacks and changes related to the air-sea exchange of greenhouse and aerosol-forming gases (CO2, N2O, DMS) are poorly understood. The main objective of GreenMelt is to investigate the links between meltwater- associated nutrient flux to coastal- and open ocean ecosystems and related consequences for net primary and export production (NPP/EP) and the air-sea fluxes of climate-relevant gases, in the Arctic Ocean around Greenland. Two key nutrients, iron (Fe) and nitrogen (N), that are crucial for phytoplankton growth, will be the prime focus of investigations. Within four interconnected workpackages, which make use of a truly synergetic high-resolution sampling approach during the GLACE expedition, we aim at (i) quantifying the contribution of meltwater to micro- and macro- nutrient inventories in the coastal- and open-ocean waters around Greenland constraining the main lithogenic sources of nutrients, specifically, Si, N, P and Fe using stable Si, Fe, Zn and radiogenic Nd isotopes. Moreover, we will (ii) assess the consequences of melt-driven changes in nutrient availability and sea-ice loss on the primary and export production and CO2 exchange, as well as the impact of sea-ice melt on pelagic ecosystem diversity, phenology, and productivity. In this context we will also study spatial changes in the emission of PP-associated dimethylsulfide (DMS). Furthermore, we propose to iii) investigate the impacts of sea-ice melting on the contribution of N2 fixation to the net community production. We will address whether iv) the marine environment around Greenland is a source of N2O, and elucidate what the main N2O producing pathway is. The synergistic, multidisciplinary approach (combining PP and N turnover rate measurements, community characterization and metagenomic analyses, trace metal and stable isotope measurements, as well as trace gas and organic particle observations), with integration of field measurements and laboratory experiments, and using an unprecedented array of instruments connected to the underway water supply, will allow us to elucidate the multifaceted impacts sea-ice melting is imposing on the productivity and biogeochemistry in polar marine environments. Results from the proposed research program will have important implications for understanding how predicted future climate change and continued sea ice retreat will ultimately affect arctic marine ecosystems and biologically-driven green house gas exchange between polar seas and the atmosphere, with important feedbacks on the global climate.

Autotrophic nitrogen (N) removal by anaerobic ammonium oxidation (anammox) is an important mechanism of fixed N elimination, both in engineered and natural systems. In wastewater treatment plants it may permit operation under energy autarky and with a better carbon footprint. However, its process control and engineering is still under development: An optimized removal process using nitritation-anammox systems must combine stable operation, high N removal efficiency and minimized greenhouse gas emissions. Yet the biogeochemical and microbiological controls on the production of N 2 O in wastewater treatment are poorly understood. Similarly, the links between system stability, activity, and microbial population shifts are not well constrained. Equally important, in natural aquatic environments, the anammox process probably plays a much greater role than it was assumed for decades, challenging the traditional perception of the natural N-cycle. The questions regarding the prime pathways of fixed N loss in natural environments (i.e., denitrification versus anammox), the controls on the balance between fixed N loss and N 2 O production, and the associated microbial population dynamics are important rationales of current research. In this applied and environmental context, an important focus of the project will be on the use of stable isotope measurements and microbiological analyses to understand and identify N metabolism in pure and mixed anammox cultures, to assess the controls on N 2 O production in nitritation-anammox systems, and to verify ties between microbial dynamics, N-turnover and optimal process control. The collaborative and highly interdisciplinary project "IsoMol", led by M. Lehmann (Unibas), seeks to understand the functioning of mixed microbial populations featuring alternative biogeochemical pathways and to characterize its dependence on environmental conditions and microbial composition. Each subproject will provide important information to improve our understanding of anammox on multiple levels (process engineering, isotope dynamics, microbiology). Subproject 1 , led by A. Joss (EAWAG) has a strong applied component, which includes the set-up of enriched and mixed culture nitritation-anammox reactors at the laboratory and pilot scales. It aims at understanding both process controls and N 2 O emission in these systems (e.g., oxygen supply and temperature), and to evaluate the benefits of anammox-based wastewater treatment with respect to conventional approaches. Sub-projects 2 and 3 , each with completely different analytical approaches, will develop and test stable isotope methods to understand and fingerprint N metabolism in pure and mixed cultures. Ammonium, nitrite and nitrate N and oxygen (O) isotope fractionation will be studied by M. Lehmann (Unibas), with a particular focus on the isotopic effect of anaerobic nitrate formation by anammox, as well as the potential of N and O isotope measurements to diagnose chemical transformations and reaction rates for different inorganic N substrates in general. Gas phase isotopic composition studies, led by J. Mohn (Empa), will identify the main processes leading to the emission of N 2 O across the range of typical operating conditions - in particular, with respect to a possible new pathway associated with the anammox metabolism. Subproject 4, led by H. Bürgmann (Eawag), will make use of cutting-edge techniques in microbiology and genetics, specifically metagenomics and metatranscriptomics, to understand the structure and function of mixed-culture anammox consortia in response to key external variables, and the mechanisms that control stability. It will apply concepts of ecological stability to the anammox process, aiming for fundamental advances in our understanding of what drives resistance and resilience of the involved microbial communities and processes. Energy-neutral municipal wastewater treatment is feasible with autotrophic N removal, but requires an optimized process design and control based on fundamental understanding of bacterial behavior and interactions. N (and O) isotope measurements in different N-species hold a strong potential to discriminate between alternative biochemical pathways and quantify reaction rates, to assess process stability and to understand the conditions that are most conducive to the emission of N 2 O. Similarly, the engineered nature of the community provides a unique level of control over what is nonetheless a complex real-life microbial community, and results will be immediately applicable to develop improvements for process engineering. In addition, the proposed research will provide the first in-depth transcriptomic analysis of anammox enrichments, and will significantly deepen our understanding of the physiological and regulatory differences between anammox strains. Our research may also contribute to the discovery and understanding of new modes of N 2 O production directly or indirectly related to anammox...