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.

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.

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...

Methane (CH 4 ) is a potent greenhouse gas with a 25 times higher global warming potential than CO 2 . Vast amounts of this gas are produced in natural wetlands and lakes. The multiple factors that control the balance between CH 4 production and consumption, and in turn regulate the emission to the atmosphere, are still not fully understood. With this proposal, we seek funding for a continuation of SNF projects 121861 and 137636, the main objectives of which were to understand the modes of, and controls on, CH 4 oxidation in the hypolimnion of eutrophic Lake Lugano. The previous efforts provided evidence for high rates of CH 4 oxidation below the oxic-anoxic interface, which was primarily attributed to micro-aerobic CH 4 oxidation within the redox-transition zone in the mid-hypolimnion. Due to this efficient biological filter, only traces of CH 4 from the sediments escape into the upper water column of the lake. However, our previous work also revealed subsurface accumulations of CH 4 at the thermocline, leading to its net emissions from the lake surface into the atmosphere (up to 4600 mol day -1 ). The "methane paradox", i.e., the persistent CH 4 supersaturation in oxic waters, was previously reported also for other lakes and the ocean, implying some unknown source of CH 4 directly in the upper water column of these environments. The C-isotopic signature of subsurface CH 4 in Lake Lugano points to biologic origin, yet the mechanisms leading to its formation remain unclear. The proposed research will aim at testing concurrent hypotheses with regards to the potential source of epilimnetic CH 4 , and at understanding the controls on the spatio-temporal dynamics of CH 4 accumulation in Lake Lugano. Combining field and laboratory measurements, along with the employment of stable isotopic, radio-label, and molecular analyses, we will test for: i) CH 4 production in association with phytoplankton productivity and anoxic microsites in sinking organic matter, ii) anaerobic CH 4 production within the digestive tracts of zooplankton, and iii) the light-induced decomposition of dissolved organic carbon (photomethanification). We will specifically investigate CH 4 production related to the exploitation (as nutrient source) and decomposition of methylated organic compounds. Some of these compounds (e.g., methylphosphonate and dimethylsulphoniopropionate) have been identified as components of phytoplankton biomass and/or metabolites during phytoplankton growth in the ocean, but their relevance in lacustrine ecosystems is unknown. Possible association between methanogens and zooplankton and/or phytoplankton aggregates will be elucidated by functional gene and lipid biomarker analysis. We will verify anticipated links between epilimnetic CH 4 accumulation, the production of methylated compounds and other substrates used during methanogenesis, as well as trophic state (i.e., nutrient availability), phytoplankton productivity and community structure. Alternative explanations for the epilimnetic CH 4 accumulations (e.g., transport of CH 4 from the littoral zone and dissolution of CH 4 bubbles) will also be examined. We hypothesize that the epilimnetic CH 4 is primarily produced in situ , and that the release of CH 4 into subsurface waters of Lake Lugano is modulated by the seasonal cycle of biological production and respiration. The proposed research will result in the first comprehensive characterization of epilimnetic CH 4 production in a deep alpine lake. It will provide a milestone 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 terrestrial and aquatic environments. Finally, established links between CH 4 production and biological productivity in the modern lake can provide the basis for temporal extrapolation. The proposed work may thus grant tools to augment our ability to predict future changes in the lacustrine CH 4 emissions

Methane (CH 4 ) is a potent greenhouse gas with a much higher global warming potential than CO 2 . A vast amount of methane is produced and stored in natural wetlands and lakes. The multiple factors that can control aerobic methane oxidation in these environments are still not fully understood. We propose an international, interdisciplinary research program between the Universities of Basel and Vienna, in which hyphenated HPLC-MS, trace-metal geochemical, isotope, biomarker and molecular microbiological techniques, applied to experimental and field samples, are combined to allow for an in-depth investigation of the role of copper (Cu) as a functional constituent of a key enzyme in bacterial methane oxidation. Of particular interest are the mechanisms of, and controls on, bacterial Cu acquisition through the release of methanobactin (MB), a Cu specific compound produced by methanotrophic bacteria to increase Cu availability and uptake. The existence of such a high affinity Cu uptake system implies that low Cu availability influences methanotrophic diversity and activity in natural environments. We propose to assess for the first time the distribution and temporal dynamics of methanobactin in two Swiss lakes, expecting new insights into the environmental controls on chalcophore production by methanotrophic bacteria and, in turn, methane oxidation under micro-aerobic conditions in lacustrine redox-transition zones. Anticipating the important role of reduced sulfur compounds in modulating Cu speciation in aquatic environments, our main goals will be to (1) address the role of sulfide as an important constraint on Cu-availability in freshwater, (2) to investigate the potential of MB exudation to increase the solubility and bioavailability of Cu- sulfides, and in turn (3) to assess whether MB production can enhance methane oxidation rates under Cu-limiting conditions in lakes. Within the frame of two PhD projects we propose the following research questions: Does Cu-availability impact and possibly limit aerobic methane oxidation in lakes? Can methanotrophic bacteria actively overcome Cu limitation through the production of methanobactin? Can we observe active methanobactin production in lakes and are there links between Cu, methanobactin concentrations, and methane oxidation rates? How does Cu/sulfide interaction influence Cu speciation in a fresh water environment, and does the kinetic stability of soluble Cu-sulfide complexes at low oxygen levels decrease the bioavailability of Cu for methanotrophs? Can Cu limitation trigger shifts of the lacustrine methanotrophic community composition? We will address these questions in a series of laboratory experiments and field measurements in the redox-transition zones of two lakes in Switzerland (Lake Lugano and Lake Cadagno). Established methods for the detection and quantification of MB will be optimized for low- concentrate analysis in the natural environment. We will search for links between Cu availability and speciation, sulfide concentrations, methanobactin production, suboxic methane oxidation rates and microbial population structure, and we will elucidate the geochemical mechanisms of bacterial Cu acquisition from sulfides. The efficiency of Cu acquisition by methanotrophic bacteria may have profound effects on the cycling of carbon and, possibly, the global climate. Furthermore, this study may be one of the starting points for research that addresses whether biochemical strategies developed by aerobic methane oxidizers to overcome Cu limitation may have been the evolutionary response to the competition for methane between anaerobic and aerobic methane oxidation.

In Böden ist die Verteilung bestimmter bakterieller Membranlipide (verzweigte Glycerol-Dialkyl-Glycerol-Tetraether - brGDGTs) abhängig von der Umgebungstemperatur und des pH-Wertes der Böden. Aufgrund von Bodenerosion vwerden diese Komponenten über Bäche und Flüsse in Seen eingetragen, wo sie sich als teil des Seesediments ablagern und als mikrobielle "Fossilien" erhalten bleiben. Die Sequenz von Seesedimenten kann mehrere hundert bis tausend, manchmal sogar zehntausende bis Millionen Jahre zurück reichen. Somit stellen brGDGTs vielversprechende Proxyindikatoren für die Rekonstruktion von Paleotemperatur und Boden pH dar und können einen wertvollen Beitrag für das Verständnis vergangener Klimavariationen in der Erdgeschichte leisten. Die Ziele des Projektes sind (i) die globale Datenbasis von brGDGTs in Böden und Seesedimenten zu erweitern, (ii) die Rolle einer möglichen aquatischen Produktion von brGDGTs in deren Ablagerung im Sedimentarchiv zu untersuchen und (iii) die kontinentale Temperatur und den Boden-pH während des Holozäns zu rekonstruieren. Für diese Ziele haben wir verschiedene Schweizer Seesedimente und Böden beprobt und die Verteilung der brGDGTs mithilfe einer verbesserten HPLC Methode analysiert. Dabei konnten wir eine neuartige brGDGT-Komponente finden (Weber et al., 2015), die auf eine bisher unterschätze Produktion von brGDGTs in lakustrinen Systemen hindeutet. Die unterschiedlichen brGDGT Quellen konnten auch in der komponentenspezifischen Zusammensetzung der stabilen Kohlenstoffisotope der brGDGTs nachgewiesen werden. Somit konnten wir zeigen, dass Isotopemessungen eine eindeutige Unterscheidung zwischen brGDGTs aquatischer und terrestrischer Herkunft ermöglichen. Für diese Projektphase haben wir vorgeschlagen, die Zusammensetzung der stabilen Kohlenstoffisotope von brGDGTs in den bereits beprobten Seesedimenten und Böden zu untersuchen um mögliche Zusammenhänge zwischen Umweltfaktoren und der lakustrinen Produktion von brGDGTs aufzudecken.