Lakes are expected to experience longer summer stratification and shorter winter mixing due to climate-induced warming. These changes will impact biogeochemical cycles, but how shifts in mixing might influence lake nitrogen removal via denitrification remains unconstrained. Here we used 15N-tracer assays, molecular techniques and flux measurements to establish the seasonal dynamics of denitrification in a eutrophic lake in Switzerland. We find that denitrification was disproportionately active during the winter mixed regime, potentially driven by a previously unrecognized chitinolytic-denitrifying microbial consortium. Moreover, denitrification was strongly governed by the relative availabilities of particulate organic carbon and nitrate. Leveraging these insights enabled accurate simulation of denitrification in a lake model, revealing that a worst-case climate scenario may shorten the mixing period by ~27 days and reduce denitrification by 8-13%, increasing nitrogen export to downstream ecosystems. We conclude that lake microbial denitrification, and its associated denitrifying consortium, will be weakened by climate change.

Abstract. Nitrous oxide (N₂O) isotopocules provide key insights into microbial nitrogen cycling, but their interpretation requires well-constrained values for both oxygen isotope signatures (δ¹⁸O–N₂O) and intramolecular ¹⁵N site preference (SP) associated with N₂O production pathways. Site preference is widely used to distinguish N₂O formation pathways because bacterial denitrification is generally assumed to yield SP values near 0 ‰ through canonical NorB-mediated NO reduction. However, the extent to which SP remains stable across physiological states and changing NO reduction pathways remains poorly constrained. Likewise, interpretation of δ¹⁸O–N₂O associated with denitrification requires understanding the relative contributions of branching kinetic isotope effects and oxygen atom exchange between nitrite and water during N₂O formation. Here, we investigated N₂O isotopic signatures during denitrification by Pseudomonas aureofaciens (NirK-bearing) and Pseudomonas chlororaphis (NirS-bearing) under active-growth and resuspension conditions using quantum cascade laser absorption spectroscopy (QCLAS) and isotope ratio mass spectrometry (IRMS). SP tracked canonical NorB-mediated NO reduction but transiently increased above +10 ‰ during early N₂O production, indicating temporary activity of alternative NO reductases. These dynamics were only resolved through continuous QCLAS measurements, highlighting the importance of time-resolved isotopic observations. While SP remains a useful indicator of NO reduction mechanisms, these results show that even within denitrification, shifts between NO reduction pathways may lead to variable SP signatures. In parallel, we quantified oxygen atom exchange between nitrite and water using incubations prepared in natural-abundance and ¹⁸O-enriched water. Contrary to expectations from denitrifier-method studies, P. aureofaciens exhibited substantial and highly variable oxygen-atom exchange (38–100 %), far exceeding previously reported values (<9 %). In contrast, P. chlororaphis showed consistently high but less variable exchange (~66 %). Resuspension experiments reproduced the characteristic low- and high-exchange behavior reported for these strains under denitrifier-method conditions, demonstrating that these exchange values are specific to the methodological framework and not representative of actively growing systems. These results show that oxygen atom exchange is not governed solely by nitrite reductase identity (NirS vs. NirK) but is strongly modulated by physiological state and metabolic context. As a result, δ¹⁸O–N₂O cannot be interpreted as a fixed tracer of denitrification pathways outside the constrained conditions of the denitrifier method. Together, these findings suggest that denitrifying bacteria may generate N₂O with a broader range of δ¹⁸O–N₂O and SP than previously assumed. This calls for a reassessment of N₂O isotopocule interpretations and emphasizes the need to integrate isotopic measurements with physiological and biochemical constraints.

The stable nitrogen (N) isotope composition of organic matter encapsulated in diatom silica frustules (δ15NDB) from sedimentary records has been used as a proxy for reconstructing N consumption dynamics in the ocean over geologic timescales. This proxy relies on the assumption that δ15NDB tracks biomass δ15N without being affected by internal N-isotope fractionation. However, recent ground-truthing efforts have shown that δ15NDB can diverge from biomass δ15N values, yet the extent of this decoupling and its drivers remain unclear.In this study, we cultured two freshwater and two marine diatom species in batch cultures to test whether δ15NDB (1) is subject to species-dependent internal 15N fractionation, and (2) reflects the δ15N of source nitrate to the same extent as biomass δ15N values, assessing potential asynchronous integration of the N isotope signal. We monitored the N-isotope systematics during diatom growth by measuring δ15N values of nitrate, bulk biomass and frustule-bound organic N throughout batch culture progression. We found that δ15NDB did not follow typical Rayleigh fractionation dynamics and remained relatively stable, while biomass δ15N increased predictably with progressive fractional nitrate consumption. The observed divergence could only be partially explained by asynchronous integration of source-nitrate δ15N values into biomass versus frustule-bound organic N (i.e., delayed incorporation into frustule-bound material), as newly formed frustules predominantly recorded the δ15N of 15N-labeled nitrate added during growth. This demonstrates that δ15NDB values primarily capture the isotopic signature of newly assimilated nitrate rather than N derived from internal, or legacy, pools. We hypothesize that shifts in growth conditions during batch culture progression alter the coupling between carbon and nitrogen metabolism, leading to physiologically driven variation in internal 15N fractionation and corresponding offsets between δ15NDB and biomass δ15N. Such sensitivity to internal isotope fractionation during biosynthesis implies that interpretation of sedimentary δ15NDB records should not only focus on changes in source inorganic δ15N, but also consider the 15N-fractionating effects related to diatom physiology and metabolism.

Nitrification is a key process in the aquatic nitrogen (N) cycle, but its products, nitrate (NO3-) and nitrous oxide (N2O), contribute to eutrophication and greenhouse gas emissions, particularly in eutrophic lakes. Variations in in-lake N cycling and N2O production pathways, as a function of seasonality and artificial oxygenation, remain poorly understood. We investigated nitrification in the artificially oxygenated eutrophic Lake Baldegg, by analyzing NO3- and N2O concentrations and isotope ratios, and measuring ammonium oxidation rates via N-15 tracer incubations over one year. An N isotope mass-balance model revealed that nitrification sustained only 5.3 +/- 0.7% of total NO3- consumption in the epilimnion, where external N loadings were influential, and considerably more in the hypolimnion (81.6 +/- 18.5%) during stratification. Dual NO3- isotope signatures (Delta delta O-18 : Delta delta N-15 ~ 1.5-1.73) and associated negative NO3- isotope anomalies confirmed epilimnetic nitrification, though external inputs partly obscured this signal. During stratification, relatively high hypolimnetic nitrification rates correlated with organic matter export, and seemed linked to sediment resuspension and artificial oxygenation. While sedimentary denitrification/DNRA dominated hypolimnetic NO3- reduction (with negligible effects on delta N-15-NO3- and delta O-18-NO3-), transient suboxic conditions enabled water column denitrification during stratification (24.1-30.2% of the total hypolimnetic denitrification). High N2O isotope site-preference values (30-35 parts per thousand) confirmed hypolimnetic ammonium oxidation as the main N2O production pathway. During winter overturn, N2O transport from the hypolimnion caused epilimnetic N2O oversaturation and atmospheric emissions up to 3.52 mu mol m(-2) d(-1). Comparison with other lakes suggests that artificial oxygenation enhances N turnover, manifesting in greater ambient N2O backgrounds and fluxes to the atmosphere.

Lakes, ponds, and reservoirs (hereafter: “lakes”) are important sources of the greenhouse gases carbon dioxide (CO2) and methane (CH4). Emissions of CO2 and CH4 from lakes are regulated in part by in-lake processes, including the production and storage of gases in the lower parts of the water column (bottom waters). However, while substantial efforts have been made to improve estimates of greenhouse gas emissions from lakes, limited data on gas concentrations along depth profiles have prevented the incorporation of bottom-water processes in global emission estimates. Here, we present GHG-depths: the largest existing dataset of depth-profile CO2 and CH4 measurements worldwide, including 522 lakes across 38 countries and all seven continents. These data include contributions from 45 research teams and 56 published studies, totaling 2558 discrete sampling events. As global change continues to alter biogeochemical cycling in lakes, these data can help improve mechanistic models to better predict greenhouse gas production and emission from lakes worldwide.

Abstract Picocyanobacteria, typically associated with oxygenated waters, thrive also in oxygen minimum zones (OMZs) and anoxic environments. The extent to which retention of diverse ancestral anaerobic traits facilitate their metabolic flexibility with regards to transitioning between oxic and anoxic habitats is poorly understood. Here, using Black Sea Cyanobium (BSA11S), we performed 13C-glucose and 15N-ammonium incubations coupled with nanoscale ion mass spectrometry (NanoSIMS) and transcriptomics to characterize the growth mode and gene expression during transition from light-to-dark and oxic-to-anoxic conditions. Incubated picocyanobacteria assimilate 13C-glucose and ammonium under anoxic-dark conditions, grow slowly without light, and retain the capability to synthesize photosynthetic pigments. They moderately upregulate genes responsible for chlorophyll-a/bilin biosynthesis, uptake of C sources, debranching of glycogen storage, and the pentose phosphate pathway for ATP, NADH, and NADPH production, as well as genes involved in NAD+ regeneration, including proton-reduction hydrogen-metabolism coupled with lactic acid fermentation. Our results support the remarkable versatility, plasticity, and potential for mixotrophy in photoautotrophs that allow them to dominate diverse aquatic systems worldwide.

Nitrate (NO3-) isotope ratios are useful indicators for nitrogen (N)-transformation processes if the associated isotope effects and their environmental controls are well-constrained. The NO3- isotope effects in natural environments may depend on the type of dissimilatory nitrate reductases involved and the degree of isotopic overprinting via NO3- regeneration. We measured the coupled N and oxygen (O) isotope effects of NO3- reduction in anoxic incubation experiments with laboratory cultures (Pseudomonas sp. and Escherichia coli) harboring different nitrate reductase enzymes (Nar and/or Nap) as well as with natural freshwater microbiomes from Lake Lugano (Switzerland) and Lake La Cruz (Spain). For comparison, isotope effects were also evaluated through coupled N and O isotope measurements in the redox transition zone of Lake Lugano North Basin. Incubation-based Rayleigh-model N isotope effects (epsilon(N)) were variable, ranging from 9 to 30 parts per thousand. In comparison, in situ epsilon(N) values (5 to 14 parts per thousand) estimated by the closed system model were lower, likely due to substrate limitation in the water column. Experiments with Pseudomonas sp. and E. coli cultures possessing Nar yielded N isotope effects of similar magnitudes and, consistent with previous data, robust Delta delta O-18:Delta delta N-15 enrichment ratios of similar to 0.9-1.0. Nitrate reduction by cultures possessing solely Nap led to lower Delta delta O-18:Delta delta N-15 of similar to 0.7. In anoxic incubations of lake water, where the effect of nitrification could be excluded, Delta delta O-18:Delta delta N-15 values between 0.6 and 1.0 suggest "community activity" of both Nar and Nap. Interestingly, stimulation of lithotrophic nitrate reduction in incubations with amended NO3- + sulfide resulted in a Delta delta O-18:Delta delta N-15 slope of 0.90 +/- 0.03 (standard error, SE), indicating Nar as the more dominant nitrate-reducing enzyme. On the contrary, stimulation of organotrophic nitrate reduction in NO3- + acetate amended incubations resulted in a significantly lower slope of 0.72 +/- 0.03 SE, suggesting a greater contribution by Nap. In contrast to the nitrate-reduction incubation experiments, the in situ Delta delta O-18:Delta delta N-15 value of 1.36 +/- 0.14 SE observed in the Lake Lugano water column was unusually high for a freshwater environment, likely reflecting the superimposed effect of NO3- production by nitrification. Our study thus underscores that both variations in activity of Nar versus Nap during nitrate reduction, as well as isotopic overprinting by nitrate regeneration may impact ecosystem NO3- isotope dynamics in natural denitrifying environments.

Organotrophic denitrification is an important nitrogen (N) removal process in lakes, but alternative N reduction processes such as lithotrophic sulfur (S)-oxidizing denitrification may be greatly underappreciated. We studied the redox transition zone (RTZ) in the meromictic water column of the North Basin of Lake Lugano (Switzerland) to characterize N transformation pathways coupled to the S and carbon (C) cycles. Incubations with N-15-labeled and unlabeled nitrate (NO3-) revealed low denitrification rates and a general limitation of organic electron donors. The most accessible fractions of exported primary production biomass may have been largely consumed in the oxic water column during sedimentation and did not reach the RTZ at ~ 100 m depth. Conversely, sulfide (H2S) and methane (CH4), major end products of anaerobic degradation of the more recalcitrant organic carbon fractions in the sediment, represent a continuous source of energy to the RTZ, fostering the establishment of a community of S- and CH4-dependent NO3- reducers, dominated by Sulfuritalea and Candidatus Methylomirabilis over several years of observation. Anoxic incubation experiments with H2S amendments revealed a strong stimulation of dissimilatory NO3- reduction to ammonium (NH4+) (DNRA), but not denitrification. High relative abundances of the archaeal NH4+ oxidizer Candidatus Nitrosopumilus and bacterial nitrifiers indicate intense NO3- regeneration by nitrification in the upper RTZ. The potential interaction between nitrification and S-driven DNRA is unclear. However, their co-occurrence suggests that, at least under conditions of carbon limitation, N recycling between the NO3- and ammonium pools predominates over N removal via complete denitrification.

Diatom frustules are well-preserved in marine and lacustrine sediments over hundreds or even thousands of years. In addition, although only in very small amounts, they also contain organic matter within their siliceous structure. Previous applications have shown that the 15N/14N ratio of the organic nitrogen contained in diatom frustules (diatom-bound δ15N, or δ15NDB) can be used as a proxy for nutrient cycling in the polar oceans, and that it is not affected by diagenetic effects. However, the applicability of this paleo-proxy to lacustrine sediments has never been tested. Here, we explore the use of δ15NDB to reconstruct the history of nitrogen dynamics in Lake Baldegg (Switzerland) over the past 300 years. This lake was heavily eutrophied due to anthropogenic activities during the 20th century, before the implementation of lake restoration measures (i.e., artificial aeration of the lake bottom since 1982). Using a multi-proxy approach (e.g., reflectance-inferred chlorophyll a and organic carbon accumulation rates, XRF sulfur counts, bulk isotopic composition, C:N ratio), we identified two distinct eutrophication phases (1880-1950 and 1950-1980) that were characterised by an increase in organic matter accumulation and primary productivity, the occurrence of bottom water anoxia, and a change in the origin of the bulk organic matter. The implementation of re-oligotrophication measures has led to the disappearance of anoxic conditions at the bottom of the lake after 1995, and a decrease in phosphorus concentrations in the lake (the latter observed in the monitoring data), which seems to have mitigated primary productivity and organic matter accumulation. δ15NDB increased during the first phase of eutrophication, which could be due to extended denitrification in the water column in an expanding anoxic water column zone, and/or limiting N concentrations for phytoplankton growth, leading to increased nitrate utilization. During the second phase, δ15NDB decreased, probably because fixed N in surface waters was no longer limiting for phytoplankton. After the implementation of re-oligotrophication measures, δ15NDB increased again, possibly the isotopic imprint of external N inputs with a high δ15N signature, such as organic fertilizers (e.g. animal manure, compost). Additionally, the δ15N of hand-picked Daphnia ephippia are lower than, and show no consistent offset to, δ15NDB, suggesting that the N isotope signal of δ15NDB is not transferred to the upper trophic level in that lake. Finally, we measured the offset between δ15NDB and δ15NBULK providing insight into the effects of early diagenesis on the N isotopic composition of bulk sediments. In Lake Baldegg, the offset reversed after the lake was artificially oxygenated, indicating a role of sediment oxygenation in the diagenetic alteration on δ15NBULK.

Potassium (both water‐soluble K + and elemental K) and levoglucosan (LG) are commonly used as chemical markers for emissions from biomass burning (BB). However, their application in complex urban environments, particularly in coal‐dominated regions, may be compromised by overlapping sources and atmospheric processes. Using a decade‐long data set (2005–2015) of rainwater chemistry and high‐resolution aerosol measurements (2018–2019) in Shanghai, China's largest megacity, this study quantitatively assesses the impacts of coal combustion on potassium and LG levels. Our analysis reveals that over 80% of K + in rainwater and ∼53% of aerosol potassium originate from coal combustion as indicated by strong correlations with industrial SO 2 emissions ( r 2 = 0.80–0.95). Similarly, LG concentrations are significantly impacted by coal combustion with molecular diagnostic ratios aligning with coal‐derived endmembers particularly during autumn. Considering the prevalence of coal‐based energy systems in most Chinese cities, we argue that pollution attributed to BB in rainwater and aerosol across urban China may be systematically overestimated when relying on currently broadly used BB tracers. By integrating long‐term observational data with receptor modeling, we further underscores the necessity of refining source attribution methodologies to better distinguish between BB and coal combustion impacts on air quality and climate in urban areas.

Abstract. Microbial methane production is a respiration reaction involved in the terminal step of anaerobic degradation of organic matter. Due to the dependency of methanogenic substrate production on fermentation reactions that produce different end productions, different sources and compositions of organic carbon (OC) may impact the methanogenic potential in lake sediments. Here, we investigate the sources and compositions of OC in sediments of Lake Geneva and how both are potentially linked to methane production. Differences in dominant long-chain fatty acid abundances and carbon isotopic compositions suggest the predominance of diagenetically altered phytoplankton-derived OC at a profundal site and temporally highly variable sources of both aquatic and terrestrial OC in a deltaic location. Despite these differences, radiotracer-based methanogenesis rate measurements and stable isotopic signatures of methane indicate significant methane production that is dominated by CO2 reduction (>95 % of total methanogenesis) in both locations. Matching this interpretation, members of well-known CO2-reducing Methanoregula sp. dominate both sites. No clear effect of OC source on methane production rates was evident. Our data demonstrate that OC of diverse sources and diagenetic states support microbial methane production, but do not indicate a clear impact of the OC source on the dominant methanogenic pathway or the community structure of methanogenic microorganisms in lacustrine sediments.

Abstract. Stable isotope analysis of O2 has emerged as a valuable tool to study O2 dynamics at various environmental scales, from molecular mechanisms to ecosystem processes. Despite its utility, there is a lack of fundamental understanding of the large variability observed in O2 isotopic fractionation at the environment- and even enzyme-level. To expand our knowledge on the potential causes of this variability, we determined 18O-kinetic isotope effects (KIEs) across a broad range of O2-consuming enzymes. The studied enzymes included nine flavin-dependent, five copper-dependent, and one copper-heme-dependent oxidases, as well as one flavin-dependent monooxygenase. For twelve of these enzymes, 18O-KIEs were determined for the first time. The comparison of 18O-KIEs, determined in this and previous studies, to calculated 18O-equilibrium isotope effects revealed distinct patterns of O-isotopic fractionation within and between enzyme groups, reflecting differences in active-site structures and O2-reduction mechanisms. Flavin-dependent O2-consuming enzymes exhibited two distinct ranges of 18O-KIEs (from 1.020 to 1.034 and from 1.046 to 1.058), likely associated with the rate-limiting steps of two different O2-reduction mechanisms (sequential vs. concomitant 2-electron transfer). In comparison, iron- and copper-dependent enzymes displayed a narrower range of 18O-KIEs, with overall lower values (from 1.009 to 1.028), which increased with the degree of O2 reduction during the rate-limiting step. Similar to flavin-dependent O2-consuming enzymes, copper-dependent O2-consuming enzymes also featured two main, yet narrower, ranges of 18O-KIEs (from 1.009 to 1.010 and from 1.017 to 1.022), likely associated with the rate-limiting formation of a copper-superoxo or copper-hydroperoxo intermediate. Overall, our findings support generalizations regarding expected 18O-KIEs ranges imparted by O2-consuming enzymes and have the potential to help interpret stable O2 isotopic fractionation patterns across different environmental scales.

Nitrous oxide (N2O), a potent greenhouse gas, primarily stems from oxidative (e.g. nitrification) and reductive (e.g., denitrification) microbial processes in aquatic and terrestrial environments. To better understand spatial and temporal N2O production, the isotopic composition of N2O, specifically 15N/14N and 18O/16O ratios, and the intramolecular distribution of 15N (i.e., site preference, SP), are typically used[1]. Distinguishing multiple concurrent processes with three isotope parameters (δ15N, δ18O, SP) remains, however, a challenge, especially in light of uncertainties regarding the isotope effects for individual processes.Here, we study the isotope effects of N2O production imparted by denitrification, focusing specifically on the intermediate step of nitrite (NO2-) reduction to nitric oxide (NO), which is catalyzed by various nitrite reductases. We study three bacterial denitrifiers: Pseudomonas chlororaphis subsp. aureofaciens, Pseudomonas chlororaphis, and Pseudomonas stutzeri. These bacteria utilize similar nitric oxide reductases (NorB) enzymes, but different nitrite reductase variants (NirS vs. NirK). We anticipate similarities in SP values, mostly controlled by NorB, but differences in δ15N-bulk and δ18O values for generated N2O, given the distinct nitrite reductase enzymes[2]. P. stutzeri strain JM300, expressing both NirS and NirK genes[3], offers a unique opportunity for studying each enzyme's distinct functions and isotopic signatures.Selected strains are incubated in batch experiments of a 0.5 L bioreactor using nitrate as a substrate. The bioreactor's headspace is continuously purged with N2. We monitor bacterial growth, NO2- concentrations, dissolved O2, pH, and temperature throughout the experiment. Simultaneously, we daily collect one sample for nitrite and nitrate N and O isotope analysis. After removing CO2 and water, N2O concentrations are monitored with Fourier-transform infrared spectroscopy. The isotopic composition of N2O is measured online using quantum-cascade-laser spectroscopy, providing real-time analysis with high precision (< 0.1 ‰). This enables real-time tracking of changes in the N and O isotope systematics (i.e., fractionation), in response to changing reaction kinetics.The preliminary data that we present will lay the basis for future investigations into the constraints on systematic heavy-isotope clumping (i.e., relative abundance of doubly substituted N2O isotopologues 15N15N16O, 14N15N18O, 15N14N18O) associated with microbial N2O production. Specifically, we will verify direct and indirect enzymatic controls (i.e., type of Nir; N-O bond equilibration with water) on the clumped-isotope abundance of 14N15N18O. [1] Toyoda, S., et al. (2017). Isotopocule analysis of biologically produced nitrous oxide in various environments. Mass Spectrometry Reviews, 36(2), 135-160. [2] Martin, T. S., et al. (2016). Nitrogen and oxygen isotopic fractionation during microbial nitrite reduction. Limnology and Oceanography, 61(3), 1134-1143. [3] Wittorf, L., et al. (2018). Expression of nirK and nirS genes in two strains of Pseudomonas stutzeri harbouring both types of NO-forming nitrite reductases. Research in microbiology, 169(6), 343-347.

While the nitrogen isotopic compositions of ammonia (δ15N-NH3) can be used to trace the NH3 origin and fate, subsequent atmospheric processes after emissions alter the δ15N signatures of initial NH3 and its aerosol products. In a "lab-in-the-field" study, we determined hourly δ15N discrimination factors (Δδ15NNH4+-NH3 = [δ15N-NH4+ - δ15N-NH3]) to reflect the combined nitrogen isotope effects during NH3 gas-to-aerosol conversion. Near a point source, the kinetic isotope effect (KIE) during the rapid neutralization of NH3(g) dominated. This resulted in 15N-depleted aerosol NH4+ (meanminmax ± 1σ = - 20.2-25.516.1 ± 3.8‰) relative to NH3 (1.6-7.29.4 ± 6.1‰), yielding a net Δδ15NNH4+-NH3 of -14.7-18.6-9.8 ± 3.2‰. Conversely, in aged ambient air, the system swiftly relaxed toward equilibrium. Bayesian modeling revealed that the equilibrium isotope effect (EIE) between NH3(aq)/NH3(g) (25 ± 12%), NH4+(aq)/NH3(aq) (34 ± 13%), and NH4+(aq/s)/NH3(g) (32 ± 15%) jointly explained over 90% of the observed Δδ15NNH4+-NH3 (20.44.431.0 ± 5.2‰). These findings suggest (i) that, in previous studies, the large variability of NH3 isotopic source signatures may partly be attributed to the differential nitrogen isotope alteration due to kinetic effects and (ii) that the inference of δ15N-NH3 signatures based on the measured δ15N-NH4+ likely leads to inaccurate NH3 source apportionment results, if only single and not the combined nitrogen isotope effects are considered.

Antimony (Sb) has gained increased attention over the past few decades due to its possible detrimental effects on biota and its potential to leach and disperse from contaminated soils. The fate of Sb in the environment is largely controlled by its chemical speciation, as well as the speciation of solid phases (e.g. Mn/Fe-oxyhydroxides) that interact with Sb in soils. Microbes have the capacity to facilitate a multitude of oxidation and reduction reactions in soils. Therefore, they exert control over the reactivity of Sb in the environment, either directly and/or indirectly, by changing Sb speciation and/or affecting the redox state of soil solid phases. Here, we outline processes that determine the behaviour of Sb in soils. We conclude that based on laboratory studies there is a good theoretical understanding of pure soil components interacting with Sb species. However, comparatively little is known concerning the contribution of these interactions in complex natural systems that are dynamic in terms of biogeochemical conditions and that can hardly be simulated using laboratory incubations. We note that important biochemical foundations of microbially driven Sb conversions (i.e. molecular constraints on organisms, genes and enzymes involved) have emerged recently. Again, these are based on laboratory incubations and investigations in environments high in Sb. In this regard, an important remaining question is which microorganisms actively impact Sb speciation under real-world conditions, in particular where Sb concentrations are low. Multiple dissolved Sb species have been described in the literature. We note that more analytical development is needed to identify and quantify possible key Sb species in natural systems, as well as anthropogenically impacted environments with only moderate Sb concentrations. With these research needs addressed, we believe that the Sb fate in the environment can be more accurately assessed, and remediation options can be developed.

Anthropogenic activities are key drivers of eutrophication and deoxygenation in coastal marine ecosystems. This stimulates the anaerobic degradation of organic matter and the release of reduced products, such as ammonium, methane, and hydrogen sulfide, which may, in turn, exacerbate eutrophication and deoxygenation. In this study, using a combination of chemical and microbial analyses, we assess the nitrogen dynamics in the water column of a eutrophic coastal system (Stockholm Archipelago) at three sites with contrasting redox conditions (oxic to long-term euxinic). At the oxic site, counter gradients of ammonium and oxygen in the water column, low nitrate δ15N values in bottom waters, and the 16S rRNA gene-based presence of nitrifiers indicate nitrification near the sediment-water interface. At the seasonally and long-term euxinic sites, nitrification, as inferred from the water column oxygen and nutrient profiles and the relative abundance of nitrifiers, primarily occurred near the oxycline. At these two sites, nitrate was removed below the oxycline through denitrification linked to sulfide oxidation by Sulfurimonas . Nitrous oxide emissions from surface waters in the archipelago reached up to 40 µmol m-2 d-1 and were not directly related to water column redox conditions, indicating that multiple factors control coastal emissions of this greenhouse gas to the atmosphere. The relative abundance of 16S rRNA genes and of N-cycle genes in metagenomes was highest at the seasonally euxinic site. Importantly, nitrifiers were significantly less abundant at the long-term euxinic site. Our results highlight that prolonged euxinia promotes recycling of ammonium over its removal, likely due to sulfide inhibition of nitrification, which sustains eutrophication and deoxygenation of coastal systems. ### Competing Interest Statement The authors have declared no competing interest.

Upwelling systems and their associated oxygen deficient zones (ODZs) are hotspots of nitrous oxide (N2O) production in the ocean. The Benguela Upwelling System (BUS) is a highly productive region and an important, yet variable source of N2O to the atmosphere. This study examined underlying processes and microbial key players governing N2O production in the BUS during the austral winter. 15N-tracer incubation experiments were conducted to track N2O production from NH4+ oxidation and denitrification. N2O production and consumption mechanisms over a longer temporal scale were determined through natural-abundance isotope analyses. Metagenomics and 16S rRNA gene amplicon sequencing were used to identify potential key prokaryotes driving N2O production. Our results showed that, compared with permanent ODZs, the BUS is characterized by a higher oxidative and a lower reductive N2O production, both of which exhibit substantial spatial variability. N2O production peaked in low-oxygen (O2) waters, with nearly equal contributions of oxidative and reductive processes, suggesting their co-occurrence across an O2 concentration range broader than previously thought. However, the observed N2O isotope signatures implied a legacy of recent and extensive N2O reduction to N2. Metagenomic and 16S rRNA gene data identified denitrifiers belonging to Thioglobaceae and the archaeal ammonia-oxidizers Nitrosopumilaceae among the potential key drivers of N2O production. Our study provides a comprehensive picture of N2O production in the BUS, revealing significant variability in the N-cycling regime and underlying N2O production mechanisms, and demonstrating the value of combining direct rate measurements with more integrative approaches, such as molecular omics and natural-abundance stable isotope tracers.

Understanding stable isotopic fractionation of dissolved O2 in aquatic environments is crucial to constrain and accurately model the processes responsible for biological O2 consumption, which are closely linked to the overall health of an ecosystem. This study aimed to investigate whether O2 consumption by microbial methane and ammonia oxidation may contribute to the observed discrepancy in O2 isotopic fractionation (18ϵ) between heterotrophic O2 respiration in laboratory incubations (-18 to -24 ‰) and in situ measurements of O2 consumption in lakes and oceans (-10 to -18 ‰). To estimate the in vivo 18ϵ values of soluble methane monooxygenase (sMMO), particulate methane monooxygenase (pMMO), and ammonia monooxygenase (AMO), which are the first enzymes required for the oxidation of methane and ammonia, experiments were performed with three methanotrophic bacteria and one comammox (complete-ammonia-oxidizing) bacterium. The resulting 18ϵ values for pMMO and AMO ranged from -18 ± 12 to -24 ± 5 ‰, not significantly different from 18ϵ values typical for heterotrophic respiration. The 18ϵ value determined for sMMO (-22 ± 2 ‰) was in the same range, yet more negative than the previously reported 18ϵ value for the isolated enzyme. Our results provide insights into the potential reaction mechanisms of pMMO and AMO and indicate that O2 consumption by sMMO, pMMO, or AMO cannot explain the observed discrepancy between in situ and laboratory 18ϵ values for "community" O2 consumption in aquatic environments. Instead, the apparent difference may be attributed to aspects involving substrate diffusion limitation.