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The isotopic composition of nitrous oxide (N2O) reveals valuable information on the biological production sources that contribute to N2O accumulation in the atmosphere, i.e. denitrification, nitrifier-denitrification and nitrification1. Isotopic fingerprints for each of these microbial pathways have been identified in past work, however, overlapping signatures of co-occurring N2O production processes2, and limitations in the robustness of associated fractionation factors under varying growth/environmental conditions3 still pose significant challenges.We will present data from the initial phase of our project, where we study N2O production and associated N and O isotopic fractionation by the denitrifier Pseudomonas aureofaciens, grown in the laboratory under different growth conditions and thus different reaction kinetics. N2O production was quantified on-line by Fourier-Transformation IR-spectroscopy (FTIR), and the isotopic composition of produced N2O was determined by quantum cascade-laser-absorption spectroscopy (QCLAS). The combination of N2O production and isotope data (continuously measured) allowed us to elucidate changes in N and O isotope fractionation in response to changing reaction kinetics.In a later phase of the project, we will expand our isotope-analytical capability by including also the doubly-substituted molecules of N2O, 15N15N16O (556), 14N15N18O (458) and 15N14N18O (548). More specifically, we will interrogate the symmetry of N – N bond formation, verify combinatorial effects during N2O production, and we will test whether Δ556, Δ458, and Δ548 (and the preference for 15N substitution in the central/terminal N-position) can be applied as proxies for reaction kinetics4, 5. 1 Yu, L., Harris, E., Lewicka Szczebak, D., Barthel, M., Blomberg, M.R., Harris, S.J., Johnson, M.S., Lehmann, M.F., Liisberg, J., Müller, C. and Ostrom, N.E., 2020. What can we learn from N2O isotope data?–Analytics, processes and modelling. Rapid Communications in Mass Spectrometry, 34(20), p.e8858.2 Kantnerová, K., Tuzson, B., Emmenegger, L., Bernasconi, S.M. and Mohn, J., 2019. Quantifying isotopic signatures of N2O using quantum cascade laser absorption spectroscopy. Chimia, 73(4), pp.232-232.3 Haslun, J.A., Ostrom, N.E., Hegg, E.L. and Ostrom, P.H., 2018. Estimation of isotope variation of N2O during denitrification by Pseudomonas aureofaciens and Pseudomonas chlororaphis: implications for N2O source apportionment. Biogeosciences, 15(12), pp.3873-3882.4 Yeung, L.Y., 2016. Combinatorial effects on clumped isotopes and their significance in biogeochemistry. Geochimica et Cosmochimica Acta, 172, pp.22-38.5 Kantnerová, K., Hattori, S., Toyoda, S., Yoshida, N., Emmenegger, L., Bernasconi, S.M. and Mohn, J., 2022. Clumped isotope signatures of nitrous oxide formed by bacterial denitrification. Geochimica et Cosmochimica Acta, 328, pp.120-129.

Aquatic sediments play a critical role in moderating the availability of fixed nitrogen (N) in the biosphere. Microbial N cycling processes, such as denitrification and anammox, contribute to fixed‑N removal as N2 gas from lakes and the ocean. N‑isotopic measurements of dissolved inorganic N (e.g., nitrate (NO3-)) can provide insights into the different sources, sinks, and pathways of N, if the associated N isotope signatures/effects are constrained. While substantial work has been done to resolve N‑loss using microbial metagenomic‑based approaches and rate measurements, how nitrogen‑loss processes imprint natural‑abundance isotopes of 15N and 18O of NO3- remains largely understudied in freshwater lake sediments. Current marine evidence suggests that water column denitrification involves high NO3- isotope effects (>20‰). In contrast, the marine NO3- isotope effect of sedimentary denitrification is suppressed at the level of the sediment‑water interface (apparent N/O isotope effect, εapp

Natural and engineered nitrogen (N) removal processes in aqueous systems represent important sources of nitrogenous gas emissions, including the potent greenhouse gas nitrous dioxide (N2O). The relevance of microbial and abiotic formation pathways can be assessed using 15N tracing techniques. While 15N-N2O analysis using optical analyzers is straightforward, quantification of 15N fractions in inorganic N compounds, ammonium (NH4+), nitrite (NO2-), and nitrate (NO3-), is typically time-consuming and labor-intensive.In this study, we developed an automated sample-preparation unit coupled to a membrane-inlet quadrupole mass spectrometer (3n-ASSP-MIMS) for the online quasi-simultaneous analysis of 15N fractions in NH4+, NO2-, and NO3-. The technique was designed and validated for applications at moderate (100 - 200 μmol L-1) to high (2 – 3 mmol L-1) N, as found in sewer systems, wastewater in treatment plants, or eutrophic surface waters, and 15N spiking (f15) between 1 and 33%.The potential of 3n-ASSP-MIMS was demonstrated in a feasibility study, where the technique, in conjunction with 15N-N2O analyses by FTIR spectroscopy, was applied to pinpoint nitrifier denitrification as the primary N2O formation pathway during partial NH4+ oxidation to NO2- in a lab-scale sequencing batch reactor.

Molecular oxygen (O2) is one of the most important electron acceptors for a large variety of biotic and abiotic processes in the environment. A wide range of oxygen isotopic fractionation associated with biological O2 consumption (e.g., respiration) has been reported in field and laboratory studies (18ɛO2 from -29 to -1 ‰). The observed variability in 18ɛO2 values has mainly been attributed to the different types of respiring organisms. But, to better understand what ultimately causes the variation in isotopic fractionation of O2, it is necessary to start investigating at the lowest level of biological complexity. All biological O2 consumption, including respiration, detoxification, and biosynthesis, occurs at the enzyme-level. A few 18ɛO2 values have been reported for isolated enzymatic O2 reduction reactions. However, these laboratory-scale studies also displayed a wide range of O-isotope effects (18ɛO2 from -33 to -10 ‰), without any systematic correlation between 18ɛO2 values and the type of enzyme, substrate, or O2-reduction mechanism. In this study, we aimed at applying O2 stable isotope analysis to a systematic selection of O2 consuming enzymes, to improve our molecular understanding of isotopic fractionation of O2 at the enzyme-level. In a first series of experiments, we have determined kinetic parameters, as well as 18ɛO2 (and 17ɛO2) values of O2 reduction for a series of copper- and flavin-dependent oxidase enzymes. O2 reduction by these oxidase enzymes occurs separately from substrate oxidation, i.e., O2 is reduced to water (four-electron reduction) or to hydrogen peroxide (two-electron reduction), independently from the type of substrate. Thus, the variability in observed O isotopic fractionation should only depend on the active-site structure and/or the O2 reduction mechanism. Our experimental 18ɛO2 values covered the same range as those previously reported for laboratory-scale studies with other enzymes. Most of the studied flavin- and copper-dependent oxidases displayed no deviation from mass-dependent fractionation (17ɛO2/18ɛO2 ≈ 0.52). We demonstrate that 18ɛO2 values systematically correlate with a given enzyme’s affinity for O2 in flavin-dependent oxidases. Furthermore, our data suggest that the range of 18ɛO2 and 17ɛO2 values differs significantly between flavin- and metal-dependent O2 consuming enzymes. These results represent an important first step towards an improved understanding and generalization of the isotopic fractionation of O2 at the enzyme- and, ultimately, at the organism-level.

Marine and lacustrine benthic habitats represent hotspots of nitrogen (N) turnover, with many N transformation processes occurring simultaneously, and at high rates. More specifically, sedimentary microbial reduction of nitrate to dinitrogen (N2), and other modes of N2 production (e.g., anammox), are the most important sinks of fixed N in aquatic environments. Natural abundance stable isotope ratio measurements can be employed to help disentangling N-loss mechanisms, given that the isotope effects (e) associated with each process are well studied.In our study, we surveyed an array of lacustrine and marine benthic environments and assessed the isotopic composition (d15N and d18O) of porewater ammonium (NH4+) and nitrate (NO3-) – the key substrates driving the various N transformation pathways, such as denitrification, anammox, nitrification and dissimilatory nitrate reduction to ammonium (DNRA). We further examined how benthic N isotope dynamics vary in different sedimentary regimes, with distinct organic matter (OM) mineralization rates. We expect the relative importance of the various N pathways to affect the NH4+ and/or NO3- isotope pools differentially.Our preliminary porewater d15N-NH4+ results suggest a diverse pattern with regard to the isotope enrichments between oligotrophic lakes, characterized by relatively strong depth gradients, and eutrophic lakes, where no significant depth gradient was observed. We hypothesize that this distinction could be attributed to different organic N availability and/or anammox contributions in the surveyed environments. Furthermore, we compared rate measurements (based on 15N addition experiments) to the N and O nitrate isotopic signatures to quantify processes, such as denitrification and DNRA. To investigate N isotope fractionation within the narrow nitracline, we employed the whole-core squeeze method, which provided a high-resolution porewater NO3-profile. Using this method, we found that the calculated community nitrate consumption e values at the nitracline (eelim_porewater) showed strong variability among the surveyed settings. We attribute such variation to shifts in the relative importance of denitrification versus DNRA and anammox between lakes of different trophic states. Overall, eelim_porewater wasconsiderably below 25‰ – the value generally reported for the biological isotope effect of denitrification (edenit).

Aerobic methane oxidation (MOx) depends critically on the availability of copper (Cu) as a crucial component of the metal centre of particulate methane monooxygenase, one of the main enzymes involved in MOx. Some methanotrophs have developed Cu acquisition strategies, in which they exude Cu-binding ligands termed chalkophores under conditions of low Cu availability. A well-characterised chalkophore is methanobactin (mb), exuded by the microaerophilic methanotroph Methylosinus trichosporium OB3b. Aerobic methanotrophs generally reside close to environmental oxic-anoxic interfaces, where the formation of Cu sulphide phases can aggravate the limitation of bioavailable Cu due to their low solubility. The reactivity of chalkophores towards such Cu sulphide mineral phases has not yet been investigated. In this study, a combination of dissolution experiments and equilibrium modelling was used to examine the dissolution and solubility of bulk and nanoparticulate Cu sulphide minerals in the presence of mb as influenced by pH, oxygen and natural organic matter. In general, we show that mb is effective at increasing the dissolved Cu concentrations in the presence of a variety of Cu sulphide phases that may potentially limit Cu bioavailability. More Cu was mobilised per mole of mb from Cu sulphide nanoparticles compared with well-crystalline bulk covellite (CuS). In general, the efficacy of mb at mobilising Cu from Cu sulphides is pH-dependent. At lower pH, e.g. pH 5, mb was ineffective at solubilizing Cu. The presence of mb increased dissolved Cu concentrations between pH 7 and 8.5, where the solubility of all Cu sulphides is generally low, both in the presence and absence of oxygen. These results suggest that chalkophore-promoted Cu mobilisation from sulphide phases is an effective extracellular mechanism for increasing dissolved Cu concentrations at oxic-anoxic interfaces, particularly in the neutral to slightly alkaline pH range. This suggests that aerobic methanotrophs may be able to fulfil their Cu requirements via the exudation of mb in natural environments where the bioavailability of Cu is constrained by very stable Cu sulphide phases., Geobiology, 20 (5), ISSN:1472-4677, ISSN:1472-4669

We investigated microbial methane oxidation in the water column of two connected but hydrodynamically contrasting basins of Lake Lugano, Switzerland. Both basins accumulate large amounts of methane in the water column below their chemoclines, but methane oxidation efficiently prevents methane from reaching surface waters. Here we show that in the meromictic North Basin water column, a substantial fraction of methane was eliminated through anaerobic methane oxidation (AOM) coupled to nitrite reduction by Candidatus Methylomirabilis. Incubations with (CH4)-C-14 and concentrated biomass from this basin showed enhanced AOM rates with nitrate (+62%) and nitrite (+43%). In the more dynamic South Basin, however, aerobic methanotrophs prevailed, Ca. Methylomirabilis was absent in the anoxic water column, and no evidence was found for nitrite-dependent AOM. Here, the duration of seasonal stratification and anoxia seems to be too short, relative to the slow growth rate of Ca. Methylomirabilis, to allow for the establishment of anaerobic methanotrophs, in spite of favorable hydrochemical conditions. Using 16 S rRNA gene sequence data covering nearly ten years of community dynamics, we show that Ca. Methylomirabilis was a permanent element of the pelagic methane filter in the North Basin, which proliferated during periods of stable water column conditions and became the dominant methanotroph in the system. Conversely, more dynamic water column conditions led to a decline of Ca. Methylomirabilis and induced blooms of the faster-growing aerobic methanotrophs Methylobacter and Crenothrix. Our data highlight that physical (mixing) processes and ecosystem stability are key drivers controlling the community composition of aerobic and anaerobic methanotrophs., The ISME Journal, 17 (5), ISSN:1751-7362, ISSN:1751-7370

The flow of Pacific water to the North Atlantic exerts a globally significant control on nutrient balances between the two ocean basins and strongly influences biological productivity in the northwest Atlantic. Nutrient ratios of nitrate (NO3-) versus phosphate (PO43-) have previously been used to complement salinity characteristics in tracing the distribution of Pacific water in the North Atlantic. We expand on this premise and demonstrate that the fraction of Pacific water as determined by NO3- : PO43- ratios can be quantitatively predicted from the isotopic composition of sub-euphotic nitrate in the northwest Atlantic. Our linear model thus provides a critically important framework for interpreting δ15N signatures incorporated into both modern marine biomass and organic material in historical and paleoceanographic archives along the northwest Atlantic margin.

Energy supply limits development through fuel constraints and climatic effects. Production of renewable energy is a central pillar of sustainability but will need to play an increasingly important role in energy generation in order to mitigate fossil-fuel based greenhouse-gas emissions. Global freshwaters represent a vast reservoir of biomass and biogenic CH4. Here we demonstrate the great potential for the optimized use of this nonfossil carbon as a source of energy that is replenishable within a human lifetime. The feasibility of up-scaled adsorption-driven technologies to capture and refine aqueous CH4 still awaits verification, yet recent estimates of global freshwater CH4 production imply that the worldwide energy demand could be satisfied by using the “biofuel” building up in lakes and wetlands. Biogenic CH4 is mostly generated from biomass produced through atmospheric CO2 uptake. Its exploitation in freshwaters can thus secure large amounts of carbon-neutral energy, helping to sustain the planetary equilibrium. ISSN:0013-936X ISSN:1520-5851

Over the past decades, fractured and karst groundwater systems have been studied intensively due to their high vulnerability to nitrate (NO3−) contamination, yet nitrogen (N) turnover processes within the recharge area are still poorly understood. This study investigated the role of the karstified recharge area in NO3− transfer and turnover by combining isotopic analysis of NO3− and nitrite (NO2−) with time series data of hydraulic heads and specific electrical conductivity from groundwater monitoring wells and a karstic spring in Germany. A large spatial variability of groundwater NO3− concentrations (0.1–0.8 mM) was observed, which cannot be explained solely by agricultural land use. Natural-abundance N and O isotope measurements of NO3− (δ15N and δ18O) confirm that NO3− derives mainly from manure or fertilizer applications. Fractional N elimination by denitrification is indicated by relatively high δ15N- and δ18O-NO3− values, elevated NO2− concentrations (0.05–0.14 mM), and δ15N-NO2− values that were systematically lower than the corresponding values of δ15N-NO3−. Hydraulic and chemical response patterns of groundwater wells suggest that rain events result in the displacement of water from transient storage compartments such as the epikarst or the fissure network of the phreatic zone. Although O2 levels of the investigated groundwaters were close to saturation, local denitrification might be promoted in microoxic or anoxic niches formed in the ferrous iron-bearing carbonate rock formations. The results revealed that (temporarily) saturated fissure networks in the phreatic zone and the epikarst may play an important role in N turnover during the recharge of fractured aquifers.

Estuaries have been identified as sources of nitrous oxide (N2O) emissions to the atmosphere but questions remain as to which production pathway(s) govern the oversaturation of N2O observed in most estuaries worldwide. Here, we use a suite of nitrate and N2O isotopes, as well as the N-15 site preference signatures of N2O to assess (1) the relative importance of different N2O production pathways in a eutrophic groundwater-impacted salt-wedge estuary and the aquifers underlying the estuary and (2) the influence of groundwater input on the overall N2O saturation in the estuary. This is one of the few studies to examine the effect of groundwater-surface water interaction on N2O cycling using N2O isotopes. The site preference values of N2O in the deep aquifer below the estuary were distinctive (83 parts per thousand +/- 25 parts per thousand) and were much higher than in either surface water (21 parts per thousand +/- 6 parts per thousand) or shallow groundwater (44 parts per thousand +/- 8 parts per thousand), suggesting the influence of multiple biotic and/or abiotic processes which proceed through multiple cycles, and/or the occurrence of a yet unidentified N2O production pathway. Isotope endmember considerations revealed that nitrifier-denitrification was the major N2O production pathway within the shallow aquifer whereas within the estuarine water column, N2O saturation was governed by chemodenitrification and discharge of N2O-laden submarine groundwater. Our study not only emphasizes the substantial, yet often underappreciated role of submarine groundwater discharge in estuarine N2O budgets, but also highlights the need to reevaluate the importance of the noncanonical denitrification pathways (i.e., chemodenitrification and nitrifier-denitrification) in controlling the overall N2O production from estuarine and groundwater environments.

N2O is a stratospheric ozone depleting substance and a potent greenhouse gas which significantly contributes to global warming. Although soils are the largest source of N2O emissions, knowledge gaps in the understanding of N2O production and reduction processes in soils still exist. Here, we investigated N2O production and consumption processes along soil depth profiles in a mesocosm experiment using natural-abundance N2O and NO3- isotopic signatures as well as abundances of soil microbial genes associated with N2O production (nirK, nirS) and reduction (nosZ). Soil columns either displayed undisturbed soil stratification (control treatments), or contained an artificial clay layer at 35 cm depth (clay treatment), which acted as a diffusion barrier and thus induced O2-limited conditions in deeper strata. We collected soil pore gas, soil solution and soil samples at five depths of the soil columns over the course of four weeks. In addition, we continuously monitored N2O fluxes at the soil surface and soil environmental parameters (oxygen, moisture, temperature) along the soil depth profiles. Microbial gene analysis in soil samples revealed similar abundances of nirK, nirS and nosZ in the two treatments across the entire soil depth profiles. The distribution of the functional genes was thus not indicative of enhanced N2O production and/or reduction in O2-limited conditions. However, lowest O2 concentrations below the clay layer were associated with highest 15N and 18O enrichments in both NO3- and N2O, indicating N2O production by denitrification and fractional N2O reduction. In addition, we found higher N2O concentrations and surface fluxes for the clay treatment. Our observations imply a dominance of N2O production over N2O reduction, even under conditions most favorable for complete denitrification.

Nitrous oxide (N2O) dominates greenhouse gas emissions in wastewater treatment plants (WWTPs). Formation of N2O occurs during biological nitrogen removal, involves multiple microbial pathways, and is typically very dynamic. Consequently, N2O mitigation strategies require an improved understanding of nitrogen transformation pathways and their modulating controls. Analyses of the nitrogen (N) and oxygen (O) isotopic composition of N2O and its substrates at natural abundance have been shown to provide valuable information on formation and reduction pathways in laboratory settings, but have never been applied to full-scale WWTPs.Here we show that N-species isotope ratio measurements at natural abundance level, combined with long-term N2O monitoring, allow identification of the N2O production pathways in a full-scale plug-flow WWTP (Hofen, Switzerland). The proposed approach can also be applied to other activated sludge systems. Heterotrophic denitrification appears as the main N2O production pathway under all tested process conditions, while nitrifier denitrification was less important, and more variable. N2O production by hydroxylamine oxidation was not observed. Fractional N2O elimination by reduction to dinitrogen (N2) during anoxic conditions was clearly indicated by a concomitant increase in SP, δ18O(N2O) and δ15N(N2O). The extent of N2O reduction correlated with the availability of dissolved inorganic N and organic substrates, which explains the link between diurnal N2O emission dynamics and organic substrate fluctuations. Consequently, dosing ammonium-rich reject water under low-organic-substrate conditions is unfavourable, as it is very likely to cause high net N2O emissions.Our results demonstrate that monitoring of the N2O isotopic composition holds a high potential to disentangle N2O formation mechanisms in engineered systems, such as full-scale WWTP. Our study serves as a starting point for advanced campaigns in the future combining isotopic technologies in WWTP with complementary approaches, such as mathematical modelling of N2O formation or microbial assays to develop efficient N2O mitigation strategies.

Anaerobic nitrate-dependent Fe(II) oxidation (NDFeO) is widespread in various aquatic environments and plays a major role in iron and nitrogen redox dynamics. However, evidence for truly enzymatic, autotrophic NDFeO remains limited, with alternative explanations involving the coupling of heterotrophic denitrification with the abiotic oxidation of structurally bound or aqueous Fe(II) by reactive intermediate nitrogen (N) species (chemodenitrification). The extent to which chemodenitrification is caused (or enhanced) by ex vivo surface catalytic effects has not been directly tested to date. To determine whether the presence of either an Fe(II)-bearing mineral or dead biomass (DB) catalyses chemodenitrification, two different sets of anoxic batch experiments were conducted: 2 mM Fe(II) was added to a low-phosphate medium, resulting in the precipitation of vivianite (Fe3(PO4)2), to which 2 mM nitrite (NO2-) was later added, with or without an autoclaved cell suspension (∼1.96×108 cells mL−1) of Shewanella oneidensis MR-1. Concentrations of nitrite (NO2-), nitrous oxide (N2O), and iron (Fe2+, Fetot) were monitored over time in both set-ups to assess the impact of Fe(II) minerals and/or DB as catalysts of chemodenitrification. In addition, the natural-abundance isotope ratios of NO2- and N2O (δ15N and δ18O) were analysed to constrain the associated isotope effects. Up to 90 % of the Fe(II) was oxidized in the presence of DB, whereas only ∼65 % of the Fe(II) was oxidized under mineral-only conditions, suggesting an overall lower reactivity of the mineral-only set-up. Similarly, the average NO2- reduction rate in the mineral-only experiments (0.004±0.003 mmol L−1 d−1) was much lower than in the experiments with both mineral and DB (0.053±0.013 mmol L−1 d−1), as was N2O production (204.02±60.29 nmol L−1 d−1). The N2O yield per mole NO2- reduced was higher in the mineral-only set-ups (4 %) than in the experiments with DB (1 %), suggesting the catalysis-dependent differential formation of NO. N-NO2- isotope ratio measurements indicated a clear difference between both experimental conditions: in contrast to the marked 15N isotope enrichment during active NO2- reduction (15εNO2=+10.3 ‰) observed in the presence of DB, NO2- loss in the mineral-only experiments exhibited only a small N isotope effect (<+1 ‰). The NO2--O isotope effect was very low in both set-ups (18εNO2 <1 ‰), which was most likely due to substantial O isotope exchange with ambient water. Moreover, under low-turnover conditions (i.e. in the mineral-only experiments as well as initially in experiments with DB), the observed NO2- isotope systematics suggest, transiently, a small inverse isotope effect (i.e. decreasing NO2- δ15N and δ18O with decreasing concentrations), which was possibly related to transitory surface complexation mechanisms. Site preference (SP) of the 15N isotopes in the linear N2O molecule for both set-ups ranged between 0 ‰ and 14 ‰, which was notably lower than the values previously reported for chemodenitrification. Our results imply that chemodenitrification is dependent on the available reactive surfaces and that the NO2- (rather than the N2O) isotope signatures may be useful for distinguishing between chemodenitrification catalysed by minerals, chemodenitrification catalysed by dead microbial biomass, and possibly true enzymatic NDFeO.

Nitrous oxide (N

Anaerobic oxidation of methane (AOM) with nitrate/nitrite as the terminal electron acceptor may play an important role in mitigating methane emissions from lacustrine environments to the atmosphere. We investigated AOM in the water column of two connected but hydrodynamically contrasting basins of a south-alpine lake in Switzerland (Lake Lugano). The North Basin is permanently stratified with year-round anoxic conditions below 120 m water depth, while the South Basin undergoes seasonal stratification with the development of bottom water anoxia during summer. We show that below the redoxcline of the North Basin a substantial fraction of methane was oxidized coupled to nitrite reduction by Candidatus Methylomirabilis. Incubation experiments with 14CH4 and concentrated biomass from showed at least 43-52%-enhanced AOM rates with added nitrate/nitrite as electron acceptor. Multiannual time series data on the population dynamics of Candidatus Methylomirabilis in the North Basin following an exceptional mixing event in 2005/2006 revealed their requirement for lasting stable low redox-conditions to establish. In the South Basin, on the other hand, we did not find molecular evidence for nitrite-dependent methane oxidizing bacteria. Our data suggest that here the dynamic mixing regime with fluctuating redox conditions is not conducive to the development of a stable population of relatively slow-growing Candidatus Methylomirabilis, despite a hydrochemical framework that seems more favorable for nitrite-dependent AOM than in the North Basin. We predict that the importance of N-dependent AOM in freshwater lakes will likely increase in future because of longer thermal stratification periods and reduced mixing caused by global warming.

We measured methane concentrations in the surface water of the northern basin of Lake Lugano in spring (May 2012) and autumn (October 2011, 2012), and calculated turbulent diffusive methane fluxes to the atmosphere. Surface water methane concentrations were highly variable in space and time but always exceeded atmospheric equilibrium. Methane concentrations were significantly lower in spring (on average 16 nmol L−1) than during the autumn sampling campaigns (on average 57 nmol L−1 in 2011 and 45 nmol L−1 in 2012). This suggests methane accumulation in the surface mixed layer during the summer productive season. The origin of the methane in the lake’s surface waters requires further assessment, but the observed concentration profiles indicate that the excess methane originates from a near-surface source, rather than from the large deep-water methane pool in the anoxic monimolimnion. As a consequence of the higher surface water methane concentrations and increased buoyancy turbulence caused by autumnal cooling of the surface boundary layer, diffusive fluxes were much higher in October (average ~97 μmol m−2 day−1, compared to 7 μmol m−2 day−1 in May 2012). The increase in methane concentration in the surface water between spring and autumn suggests links between methane accumulation and the annual biological cycle, yet seasonal changes in wind and temperature forcing of methane emission likely play an important modulating role. While the relative importance of biological versus physical controls on methane emission in Lake Lugano awaits further investigations, our study underscores that lakes can act as an important source of methane to the atmosphere, even when the lake-internal microbial methane filter in the water column seems to work efficiently.

Pockmarks are crater-like depressions formed by upward fluid flow (gas and/or liquid) through the unconsolidated sediment column on the floor of oceans and lakes. While pockmarks are well described in the marine realm, they have essentially been overlooked in lacustrine settings, likely due to a lack in economic interest to apply high-resolution hydroacoustic techniques in lakes. A swath-bathymetry survey on Lake Thun, Switzerland, revealed the existence of three pockmark systems. One pockmark (110 m in diameter) was discovered near a big karst system at Beatenberg at a water depth of similar to 217 m. Its activity is probably associated with episodic groundwater seepage induced by earthquakes, floods and snowmelt. At another site, Daerligen, we detected at similar to 60 m water depth the presence of multiple smaller pockmarks (similar to 1.5 to 10 m in diameter) that seem to be active, continuously liberating CH4 gas by bubble ebullition. The CH4 displayed a biogenic carbon isotopic signature, however, the exact origin of the gas remains unknown. The third site, Tannmoos (similar to 35 m water depth), comprises two large pockmarks (20-43 m in diameter) connected to a karst system in gypsum-carrying bedrock. One of these pockmarks is constituted of several unit pockmarks (e.g., sub-pockmarks; 0.3 to 0.8 m in diameter). While strong evidence is still lacking, we suggest that groundwater discharge occasionally occurs through these unit pockmarks during periods of intense precipitation. Hence, this study reveals the existence of three pockmark systems of variable morphology and mechanisms of formation within the same lake, reflecting different hydrological and biogeochemical regimes. Moreover, it underscores the potential importance of pockmarks in influencing hydrological and CH4 budgets in lakes. Clearly more work on quantifying seasonal fluxes of groundwater and CH4 release via lacustrine pockmarks is required, and it needs to be seen whether the observations made in Lake Thun are universal and apply also to many other lacustrine environments worldwide., Frontiers in Water, 3, ISSN:2624-9375

Mainstream anaerobic ammonium oxidation (anammox) represents one of the most promising energy-efficient mechanisms of fixed nitrogen elimination from wastewaters. However, little is known about the exact processes and drivers of microbial community assembly within the complex microbial biofilms that support anammox in engineered ecosystems. Here, we followed anammox biofilm development on fresh carriers in an established 8m3 mainstream anammox reactor that is exposed to seasonal temperature changes (~25-12°C) and varying NH4+ concentrations (5-25 mg/L). We use fluorescence in situ hybridization and 16S rRNA gene sequencing to show that three distinct stages of biofilm development emerge naturally from microbial community composition and biofilm structure. Neutral modelling and network analysis are employed to elucidate the relative importance of stochastic versus deterministic processes and synergistic and antagonistic interactions in the biofilms during their development. We find that the different phases are characterized by a dynamic succession and an interplay of both stochastic and deterministic processes. The observed growth stages (Colonization, Succession and Maturation) appear to be the prerequisite for the anticipated growth of anammox bacteria and for reaching a biofilm community structure that supports the desired metabolic and functional capacities observed for biofilm carriers already present in the system (~100gNH4-N m3 d−1). We discuss the relevance of this improved understanding of anammox-community ecology and biofilm development in the context of its practical application in the start-up, configuration, and optimization of anammox biofilm reactors., Water Research, 200, ISSN:0043-1354, ISSN:1879-2448

Ammonia (NH3) is an important agent involved in atmospheric chemistry and nitrogen cycling. Current estimates of NH3 emissions from biomass burning (BB) differ by more than a factor of 2, impeding a reliable assessment of their environmental consequences. Combining highresolution satellite observations of NH3 columns with network measurements of the concentration and stable nitrogen isotope composition (δ15N) of NH3, we present coherent estimates of the amount of NH3 derived from BB in the heartland of Southeast Asia, a tropical monsoon environment. Our results reveal a strong variability in atmospheric NH3 levels in time and space across different landscapes. All of the evidence on hand suggests that anthropogenic activities are the most important modulating control with respect to the observed patterns of NH3 distribution in the study area. Nisotope balance considerations revealed that during the intensive fire period, the atmospheric input from BB accounts for no more than 21 ± 5 % (1s) of the ambient NH3, even at the rural sites and in the proximity of burning areas. Our N-isotope-based assessment of the variation in the relative contribution of BB-derived NH3 is further validated independently through the measurements of particulate KC, a chemical tracer of BB. Our findings underscore that BB-induced NH3 emissions in tropical monsoon environments can be much lower than previously anticipated, with important implications for future modeling studies to better constrain the climate and air quality effects of wildfires., SCOPUS: ar.j, info:eu-repo/semantics/published

Identifying sources of anthropogenic pollution, and assessing the fate and residence time of pollutants in aquifers is important for the management of groundwater resources, and the ecological health of groundwater dependent ecosystems. This study investigates anthropogenic contamination in the shallow alluvial aquifer of the Marana-Casinca, hydraulically connected to the Biguglia lagoon (Corsica, France). A multi-tracer approach, combining geochemical and environmental isotopic data (δ18O-H2O, δ2H-H2O, 3H, δ15N-NO3−, δ18O-NO3−, δ11B), and groundwater residence-time tracers (3H and CFCs) was carried out in 2016, and integrated with a study of land use evolution in the catchment during the last century. Groundwater NO3− concentrations, ranged between 2 mg/L and up to 30 mg/L, displaying the degradation of groundwater quality induced by anthropogenic activities (agricultural activities). Comparatively high δ15N-NO3− values (up to 19.7‰) in combination with δ11B values that were significantly lower (between 23‰ and 26‰) than the seawater background are indicative of sewage contamination. The ongoing deterioration of groundwater quality can be attributed to the uncontrolled urbanization development all over the alluvial plain, with numerous sewage leakages from the sanitation network and private sewage systems. Integration of contaminant and water-residence time data revealed a progressive accumulation of pollutants with time in the groundwater, particularly in areas with major anthropogenic pressure and slow dynamic groundwater flow. Our approach provides time-dependent insight into nitrogen pollution in the studied aquifer over the past decades, revealing a systematic change in the dominant NO3− source, from agricultural to sewage contamination. Yet, today's low groundwater quality is to large parts due to legacy pollution from land-use practices several decades ago, underlining the poor self-remediating capacity of this hydrosystem. Our results can be taken as warning that groundwater pollution that happened in the recent past, or today, may have dire impacts on the quality of groundwater-dependent ecosystems in the future.

Small boreal lakes are known to contribute significantly to global CH4 emissions. Lake Lovojärvi is a eutrophic lake in southern Finland with bottom water CH4 concentrations up to 2 mM. However, the surface water concentration, and thus the diffusive emission potential, was low (, Biogeosciences, 18 (10), ISSN:1726-4170

Anaerobic ammonium oxidation (anammox) plays an important role in aquatic systems as a sink of bioavailable nitrogen (N), and in engineered processes by removing ammonium from wastewater. The isotope effects anammox imparts in the N isotope signatures (15N/14N) of ammonium, nitrite, and nitrate can be used to estimate its role in environmental settings, to describe physiological and ecological variations in the anammox process, and possibly to optimize anammox-based wastewater treatment. We measured the stable N-isotope composition of ammonium, nitrite, and nitrate in wastewater cultivations of anammox bacteria. We find that the N isotope enrichment factor 15ε for the reduction of nitrite to N2 is consistent across all experimental conditions (13.5‰ ± 3.7‰), suggesting it reflects the composition of the anammox bacteria community. Values of 15ε for the oxidation of nitrite to nitrate (inverse isotope effect, − 16 to − 43‰) and for the reduction of ammonium to N2 (normal isotope effect, 19–32‰) are more variable, and likely controlled by experimental conditions. We argue that the variations in the isotope effects can be tied to the metabolism and physiology of anammox bacteria, and that the broad range of isotope effects observed for anammox introduces complications for analyzing N-isotope mass balances in natural systems., Scientific Reports, 11 (1), ISSN:2045-2322

We conducted a 15N tracer experiment in laboratory microcosms with field-fresh soil samples from a biodiversity experiment to evaluate the relationship between grassland biodiversity and N cycling. To embrace the complexity of the N cycle, we determined N exchange between five soil N pools (labile and recalcitrant organic N, dissolved NH4+ and NO3− in soil solution, and exchangeable NH4+) and eight N transformations (gross N mineralization from labile and recalcitrant organic N, NH4+ immobilization into labile and recalcitrant organic N, autotrophic nitrification, heterotrophic nitrification, NO3− immobilization, adsorption of NH4+) expected in aerobic soils with the help of the N-cycle model Ntrace. We used grassland soil of the Jena Experiment, which includes plant mixtures with 1 to 60 species and 1 to 4 functional groups (legumes, grasses, tall herbs, small herbs). The 19 soil samples of one block of the Jena Experiment were labeled with either 15NH4+ or 15NO3- or both. In the presence of legumes, gross N mineralization and autotrophic nitrification increased significantly because of higher soil N concentrations in legume-containing plots and high microbial activity. Similarly, the presence of grasses significantly increased the soil NH4+ pool, gross N mineralization, and NH4+ immobilization, likely because of enhanced microbial biomass and activity by providing large amounts of rhizodeposits through their dense root systems. In our experiment, previously reported plant species richness effects on the N cycle, observed in a larger-scale field experiment within the Jena Experiment, were not seen. However, specific plant functional groups had a significant positive impact on the N cycling in the incubated soil samples.

The isotopic composition of nitrous oxide (N2 O) provides useful information for evaluating N2 O sources and budgets. Due to the co-occurrence of multiple N2 O transformation pathways, it is, however, challenging to use isotopic information to quantify the contribution of distinct processes across variable spatiotemporal scales. Here, we present an overview of recent progress in N2 O isotopic studies and provide suggestions for future research, mainly focusing on: analytical techniques; production and consumption processes; and interpretation and modelling approaches. Comparing isotope-ratio mass spectrometry (IRMS) with laser absorption spectroscopy (LAS), we conclude that IRMS is a precise technique for laboratory analysis of N2 O isotopes, while LAS is more suitable for in situ/inline studies and offers advantages for site-specific analyses. When reviewing the link between the N2 O isotopic composition and underlying mechanisms/processes, we find that, at the molecular scale, the specific enzymes and mechanisms involved determine isotopic fractionation effects. In contrast, at plot-to-global scales, mixing of N2 O derived from different processes and their isotopic variability must be considered. We also find that dual isotope plots are effective for semi-quantitative attribution of co-occurring N2 O production and reduction processes. More recently, process-based N2 O isotopic models have been developed for natural abundance and 15 N-tracing studies, and have been shown to be effective, particularly for data with adequate temporal resolution. Despite the significant progress made over the last decade, there is still great need and potential for future work, including development of analytical techniques, reference materials and inter-laboratory comparisons, further exploration of N2 O formation and destruction mechanisms, more observations across scales, and design and validation of interpretation and modelling approaches. Synthesizing all these efforts, we are confident that the N2 O isotope community will continue to advance our understanding of N2 O transformation processes in all spheres of the Earth, and in turn to gain improved constraints on regional and global budgets.

It is a puzzle as to why more severe haze formed during the New Year Holiday in 2020 (NYH‐20), when China was in an unprecedented state of shutdown to contain the coronavirus (COVID‐19) outbreak, than in 2019 (NYH‐19). We performed a comprehensive measurement and modeling analysis of the aerosol chemistry and physics at multiple sites in China (mainly in Shanghai) before, during, and after NYH‐19 and NYH‐20. Much higher secondary aerosol fraction in PM2.5 were observed during NYH‐20 (73%) than during NYH‐19 (59%). During NYH‐20, PM2.5 levels correlated significantly with the oxidation ratio of nitrogen (r 2 = 0.77, p < 0.01), and aged particles from northern China were found to impede atmospheric new particle formation and growth in Shanghai. A markedly enhanced efficiency of nitrate aerosol formation was observed along the transport pathways during NYH‐20, despite the overall low atmospheric NO2 levels., Key Points Higher concentrations and distinct compositions of aerosol particles were observed during the COVID‐19 shutdownFast formation of secondary inorganic aerosol contributed to high aerosol mass loadingLonger‐range, regional transport facilitated and enhanced particulate nitrate formation

Pockmarks are crater-like depressions on the floor of oceans and lakes formed by the upward transport of fluids through the unconsolidated sediment column. The fluid flow through marine pockmarks is considered to enhance hydrological and biogeochemical exchanges between the sediments and the water body. While a similar relevance can be expected in lakes, the importance of lacustrine pockmarks in this regard is virtually unexplored.Lake Thun (48.3 km2 surface area), Switzerland, is an excellent system to study lacustrine pockmarks as it exhibits several sites with different geological and biogeochemical settings. One of the pockmark sites is characterized by evident methane (CH4) ebullition and high CH4 concentrations from ~2.4 to 8.9 mM within the sediments beneath. A large pockmark with a diameter of 110 m is located adjacent to the rock wall of a karst system and might thus be associated with groundwater discharge into the lake. Finally, spikes in electrical conductivity detected during a survey with a remotely operated vehicle (ROV) at a third pockmark site suggest a hydrogeological connection with the groundwater system in the underlying Triassic bedrock.This third pockmark site we are studying more closely. We observed that the sediments inside the pockmark were clearly more liquified as compared to those at a reference site (outside the pockmark), providing further evidence for groundwater discharge that might presently be active. The porewater chemistry was similar at the two sites, except for the total dissolved Fe concentration which was about 2 to 5-fold lower inside the pockmark than at a reference site. Further chemical analysis of porewaters and the water column above the pockmark as well as a molecular investigation (e.g. 16S rRNA) of the sediments will be performed at two different seasons of the year (in fall and spring during the snowmelt season). All together, these results should help us to better assess the influence of groundwater discharge via this pockmark site on the hydrological balance and on the biogeochemistry of the lake, as well as to expand our limited knowledge on the mechanism of lacustrine pockmarks in general.