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Nitrous oxide (N2O) is the third most important long-lived greenhouse gas and agriculture is the largest source of N2O emissions. Curbing N2O emissions requires understanding influences on the flux and sources of N2O. We measured flux and evaluated microbial sources of N2O using site preference (SP; the intramolecular distribution of 15N in N2O) in flux chambers from a grassland tilling and agricultural fertilization experiments and atmosphere. We identified values greater than that of the average atmosphere to reflect nitrification and/or fungal denitrification and those lower than atmosphere as increased denitrification. Our spectroscopic approach was based on an extensive calibration with 18 standards that yielded SP accuracy and reproducibility of 0.7 ‰ and 1.0 ‰, respectively, without preconcentration. Chamber samples from the tilling experiment taken ~ monthly over a year showed a wide range in N2O flux (0–1.9 g N2O-N ha−1 d−1) and SP (− 1.8 to 25.1 ‰). Flux and SP were not influenced by tilling but responded to sampling date. Large fluxes occurred in October and May in no-till when soils were warm and moist and during a spring thaw, an event likely representing release of N2O accumulated under snow cover. These high fluxes could not be ascribed to a single microbial process as SP differed among chambers. However, the year-long SP and flux data for no-till showed a slight direct relationship suggesting that nitrification increased with flux. The comparative data in till showed an inverse relationship indicating that high flux events are driven by denitrification. Corn (Zea mays) showed high fluxes and SP values indicative of nitrification ~ 4 wk after fertilization with subsequent declines in SP indicating denitrification. Although there was no effect of fertilizer treatment on flux or SP in switchgrass (Panicum virgatum), high fluxes occurred ~ 1 month after fertilization. In both treatments, SP was indicative of denitrification in many instances, but evidence of nitrification/fungal denitrification also prevailed. At 2 m atmospheric N2O SP had a range of 31.1 ‰ and 14.6 ‰ in the grassland tilling and agricultural fertilization experiments, respectively. These data suggest the influence of soil microbial processes on atmospheric N2O and argue against the use of the global average atmospheric SP in isotopic modeling approaches.

Atmospheric nitrous oxide (N2O) is a potent greenhouse gas thought to be mainly derived from microbial metabolism as part of the denitrification pathway. Here, we report that in unexplored peat soils of Central and South America, N2O production can be driven by abiotic reactions (≤ 98 %) highly competitive to their enzymatic counterparts. Extracted soil iron positively correlated with in-situ abiotic N2O production determined by isotopic tracers. Moreover, we found that microbial N2O reduction accompanied abiotic production, essentially closing a coupled abiotic-biotic N2O cycle. Anaerobic N2O consumption occurred ubiquitously (pH 6.4-3.7), with proportions of diverse clade II N2O-reducers increasing with consumption rates. Our findings show denitrification in tropical peat soils is not a purely biological process, but rather a “mosaic” of abiotic and biotic reduction reactions. We predict hydrological and temperature fluctuations differentially affect abiotic and biotic drivers and further contribute to the high N2O flux variation in the region.

© 2020 The Authors. This article is licensed under a Creative Com- mons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any med- ium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Laurentian Great Lakes have been subject to substantial modification from diverse anthropogenic stressors, including nutrient enrichment, climate change, chemical and biological pollutants, and invasive species, yet little is known of the relative historical influence of these factors. Here we analyze diverse fossil metrics from vibracores at two sites, a bay area (Anchor Bay) and a tributary (Clinton River) in the Lake St. Clair ecosystem to determine the ecological responses from land-use practices and invasive mussel invasions. Sediment cores spanning over 100 years indicated that the expansion of non-native Dreissena polymorpha and Dreissena rostriformis (dreissenid mussels) into Anchor Bay site after the mid-1990s was associated with ~ 60 to 95% reduction in algal and cyanobacterial abundances and twofold increase in sedimentary organic matter (SOM) and bioavailable phosphorus. These increases in SOM and bioavailable phosphorus are relatively similar to increases inferred from the late nineteenth century when large portions of the watershed were cleared and drained for agriculture. In contrast, the Clinton River site experienced a continuous increase in the influx of nutrients, organic matter, and elevated sedimentary phototrophic pigments during the twentieth century and into the twenty-first century. Site comparisons suggest different mechanisms inducing changes in primary production varied, where Anchor Bay was mainly affected by the comparatively recent (since ca. mid-1990s) endogenous influence of invasive species, while the Clinton River site was primarily influenced by the input of exogenous anthropogenic nutrients over the past 100 years. These new findings illustrate that watershed management and policies within large lakes with multi-jurisdictional (national) Area of Concerns should consider site-specific regulatory mechanisms. This work was supported in part by the National Science Foundation under Award No. EAR-1039122, NSERC Canada, Canada Foundation for Innovation, Province 123 190 J Paleolimnol (2020) 63:177–193 of Saskatchewan, University of Regina, Queen’s University Belfast, and Canada Research Chair funding. Faculty yes

Freshwaters are a significant yet understudied component of the global nitrous oxide (N2O) budget. Despite the potential importance of the Laurentian Great Lakes in the freshwater N2O budget, studies have been extremely limited to date. This study evaluated the production pathways, concentrations, and atmospheric emissions of N2O across the two trophic extremes of the Great Lakes: the deep oligotrophic waters of Lake Superior and shallow eutrophic zones of western Lake Erie. Production pathways via denitrification and nitrification were evaluated through stable isotope analysis, and atmospheric emissions were determined from surface concentrations and wind speed. Across all sites and dates, N2O saturation ranged from 98 to 129% in Lake Superior and 93 to 676% in Sandusky Bay, Lake Erie, indicating these lakes are net sources of N2O to the atmosphere. Isotopic site preference values (SP) suggest a mix of production pathways, with nitrification dominating most time periods and denitrification occurring under conditions of high nutrient availability and microbial activity. N2O atmospheric emissions were strong but highly variable in Lake Erie, and emissions in Lake Superior were consistently low (− 0.26 to 33.03 and − 0.14 to 1.41 μmol N m−2 day−1, respectively). Our findings highlight two paradigms of N2O production and emissions: low, wind-driven rates in deep oligotrophic zones and temporally dynamic rates driven by N loading in shallow eutrophic zones. Offshore regions likely make up the majority of the N2O budget for the Great Lakes, yet nearshore regions have a greater capacity for increased N2O emissions in the face of increased nutrient loading.

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.

[Image: see text] We describe an approach for determining biological N(2) production in soils based on the proportions of naturally occurring (15)N(15)N in N(2). Laboratory incubation experiments reveal that biological N(2) production, whether by denitrification or anaerobic ammonia oxidation, yields proportions of (15)N(15)N in N(2) that are within 1‰ of that predicted for a random distribution of (15)N and (14)N atoms. This relatively invariant isotopic signature contrasts with that of the atmosphere, which has (15)N(15)N proportions in excess of the random distribution by 19.1 ± 0.1‰. Depth profiles of gases in agricultural soils from the Kellogg Biological Station Long-Term Ecological Research site show biological N(2) accumulation that accounts for up to 1.6% of the soil N(2). One-dimensional reaction-diffusion modeling of these soil profiles suggests that subsurface N(2) pulses leading to surface emission rates as low as 0.3 mmol N(2) m(–2) d(–1) can be detected with current analytical precision, decoupled from N(2)O production.

Soil microbial processes, stimulated by agricultural fertilization, account for 90 % of anthropogenic nitrous oxide ( N2O ), the leading source of ozone depletion and a potent greenhouse gas. Efforts to reduce N2O flux commonly focus on reducing fertilization rates. Management of microbial processes responsible for N2O production may also be used to reduce N2O emissions, but this requires knowledge of the prevailing process. To this end, stable isotopes of N2O have been applied to differentiate N2O produced by nitrification and denitrification. To better understand the factors contributing to isotopic variation during denitrification, we characterized the δ15 N, δ18 O and site preference (SP; the intramolecular distribution of 15N in N2O ) of N2O produced during NO 3 - reduction by Pseudomonas chlororaphis subsp. aureofaciens and P. c. subsp. chlororaphis. The terminal product of denitrification for these two species is N2O because they lack the gene nitrous oxide reductase, which is responsible for the reduction of N2O to N 2 . In addition to species, treatments included electron donor (citrate and succinate) and electron donor concentration (0.01, 0.1, 1 and 10 mM) as factors. In contrast to the expectation of a Rayleigh model, all treatments exhibited curvilinear behaviour between δ15 N or δ18 O and the extent of the reaction. The curvilinear behaviour indicates that the fractionation factor changed over the course of the reaction, something that is not unexpected for a multi-step process such as denitrification. Using the derivative of the equation, we estimated that the net isotope effects ( η ) vary by as much as 100 ‰ over the course of a single reaction, presenting challenges for using δ15 N and δ18 O as apportionment tools. In contrast, SP for denitrification was not affected by the extent of the reaction, the electron donor source or concentration, although the mean SP of N2O produced by each species differed. Therefore, SP remains a robust indicator of the origin of N2O . To improve apportionment estimates with SP, future studies could evaluate other factors that contribute to the variation in SP.

The Laurentian Great Lakes (LGL) constitute one of the largest freshwater systems in the world while providing social and economic value to two powerful nations. The spatial scale of these inland seas falls between two endpoints: small lakes and oceans. Lacustrine in many characteristics, the LGL often require a scientific approach with attributes similar to those of oceanography. There is a strong sense that within the LGL support for scientific research has not kept pace with the need for process-oriented research and that we lack basic information needed to forecast change, mitigate impacts and restore and preserve the LGL. Consequently, 58 researchers met in September 2014 and identified five “Grand Challenges for Research in the LGL”: (1) How has this vast inland freshwater system responded to shifting climate in the past, and how will it respond in the future? (2) What is the current status of the most important ecosystem processes, including their variability in space and time? (3) What processes are characteristic only of large lakes, and how do the distinct habitats integrate into a whole? (4) What are the ecosystem responses to major anthropogenic forces such as nutrients and invasive species, and are these reversible? and (5) What are the small to large-scale linkages and feedbacks among societal decisions, biological systems, and physicochemical dynamics? An urgent need exists for a unified scientific voice that articulates the Grand Challenges for research in the LGL and the need for associated funding. This treatise describing the Grand Challenges develops that voice.

N2O is a highly potent greenhouse gas and arable soils represent its major anthropogenic source. Field-scale assessments and predictions of soil N2O emission remain uncertain and imprecise due to the episodic and microscale nature of microbial N2O production, most of which occurs within very small discrete soil volumes. Such hotspots of N2O production are often associated with decomposing plant residue. Here we quantify physical and hydrological soil characteristics that lead to strikingly accelerated N2O emissions in plant residue-induced hotspots. Results reveal a mechanism for microscale N2O emissions: water absorption by plant residue that creates unique micro-environmental conditions, markedly different from those of the bulk soil. Moisture levels within plant residue exceeded those of bulk soil by 4–10-fold and led to accelerated N2O production via microbial denitrification. The presence of large (O >35 μm) pores was a prerequisite for maximized hotspot N2O production and for subsequent diffusion to the atmosphere. Understanding and modelling hotspot microscale physical and hydrologic characteristics is a promising route to predict N2O emissions and thus to develop effective mitigation strategies and estimate global fluxes in a changing environment. Production of the greenhouse gas nitrous oxide occurs episodically in small soil volumes. Soil microcosm experiments reveal that water absorption by plant residue raises moisture levels and accelerates nitrous oxide production by microbial denitrification.

Iron redox cycling in metal-rich, hypersaline, anoxic brines plays a central role in the biogeochemical evolution of life on Earth, and similar brines with the potential to harbor life are thought to exist elsewhere in the solar system. To investigate iron biogeochemical cycling in a terrestrial analog we determined the iron redox chemistry and isotopic signatures in the cryoencapsulated liquid brines found in frozen Lake Vida, East Antarctica. We used both in situ voltammetry and the spectrophotometric ferrozine method to determine iron speciation in Lake Vida brine (LVBr). Our results show that iron speciation in the anoxic LVBr was, unexpectedly, not free Fe(II). Iron isotope analysis revealed highly depleted values of −2.5‰ for the ferric iron of LVBr that are similar to iron isotopic signatures of Fe(II) produced by dissimilatory iron reduction. The presence of Fe(III) in LVBr therefore indicates dynamic iron redox cycling beyond iron reduction. Furthermore, extremely low δ18O–SO42− values (−9.7‰) support microbial iron-sulfur cycling reactions. In combination with evidence for chemodenitrification resulting in iron oxidation, we conclude that coupled abiotic and biotic redox reactions are driving the iron cycle in Lake Vida brine. Our findings challenge the current state of knowledge of anoxic brine chemistry and may serve as an analogue for icy brines found in the outer reaches of the solar system.

Coral communities of the Florida Reef Tract (FRT) have changed dramatically over the past 30 years. Coral cover throughout the FRT is disproportionately distributed, &gt, 70% of total coral cover is found within the inshore patch reef zone (&lt, 2 km from shore) compared to 30% found within the offshore bank reef zone (&gt, 5 km from shore). Coral mortality from disease has been differentially observed between inshore and offshore reefs along the FRT. Therefore, differences between the response of inshore and offshore coral populations to bacterial challenge may contribute to differences in coral cover. We examined immune system activation in Porites astreoides (Lamarck, 1816), a species common in both inshore and offshore reef environments in the FRT. Colonies from a representative inshore and offshore site were reciprocally transplanted and the expression of three genes monitored biannually for two years (two summer and two winter periods). Variation in the expression of eukaryotic translation initiation factor 3, subunit H (eIF3H), an indicator of cellular stress in Porites astreoides, did not follow annual patterns of seawater temperatures (SWT) indicating the contribution of other stressors (e.g., irradiance). Greater expression of tumor necrosis factor (TNF) receptor associated factor 3 (TRAF3), a signaling protein of the inflammatory response, was observed among corals transplanted to, or located within the offshore environment indicating that an increased immune response is associated with offshore coral more so than the inshore coral (p &lt, 0.001). Corals collected from the offshore site also upregulated the expression of adenylyl cyclase associated protein 2 (ACAP2), increases which are associated with decreasing innate immune system inflammatory responses, indicating a counteractive response to increased stimulation of the innate immune system. Activation of the innate immune system is a metabolically costly survival strategy. Among the two reefs studied, the offshore population had a smaller mean colony size and decreased colony abundance compared to the inshore site. This correlation suggests that tradeoffs may exist between the activation of the innate immune system and survival and growth. Consequently, immune system activation may contribute to coral community dynamics and declines along the FRT.

Many lacustrine systems, despite management efforts to control eutrophication, are hypoxic during stratified periods. Hypoxia is a major concern, not only for its impact on aquatic life but also for its potential to stimulate production of the greenhouse gases, methane (CH4) and nitrous oxide (N2O). We investigated the drivers of hypoxia in Muskegon Lake, a temperate dimictic freshwater estuary that experiences frequent hypolimnetic mixing due to atmospheric forces, riverine inputs, and intrusion of oxic water from coastal upwelling in Lake Michigan. Primary production and respiration (R) rates obtained from a δ18O mass balance model were similar to other mesotrophic environments (0.56–26.31 and 0.57–13.15 mmol O2 m−3 day−1, respectively), although high P/R (≥2 in mid-summer) indicated there is sufficient autochthonous production to support hypoxia development and persistence. The isotopic enrichment factor for respiration (eobs) varied markedly and was least negative in August of both sampling years, consistent with high R rates. Hypoxic conditions were associated with accumulation of N2O but not CH4, and emissions of N2O are among the highest reported from lakes. The average N2O site preference value of 25.4‰ indicates that the majority of N2O was produced by nitrification via hydroxylamine oxidation, despite the presence of resilient hypoxia. While it has been hypothesized that denitrification acts as a sink for N2O in hypoxic lakes, it is clear that Muskegon Lake functions as a strong source of N2O via nitrification. Further considerations of lakes as global sources of N2O thus warrant a closer evaluation of nitrification-fueled N2O production.

Lake Vida, in the Victoria Valley of East Antarctica, is frozen, yet harbors liquid brine (~20% salt, >6 times seawater) intercalated in the ice below 16 m. The brine has been isolated from the surface for several thousand years. The brine conditions (permanently dark, -13.4 °C, lack of O2 , and pH of 6.2) and geochemistry are highly unusual. For example, nitrous oxide (N2 O) is present at a concentration among the highest reported for an aquatic environment. Only a minor 17 O anomaly was observed in N2 O, indicating that this gas was predominantly formed in the lake. In contrast, the 17 O anomaly in nitrate (NO3-) in Lake Vida brine indicates that approximately half or more of the NO3- present is derived from atmospheric deposition. Lake Vida brine was incubated in the presence of 15 N-enriched substrates for 40 days. We did not detect microbial nitrification, dissimilatory reduction of NO3- to ammonium (NH4+), anaerobic ammonium oxidation, or denitrification of N2 O under the conditions tested. In the presence of 15 N-enriched nitrite (NO2-), both N2 and N2 O exhibited substantial 15 N enrichments; however, isotopic enrichment declined with time, which is unexpected. Additions of 15 N-NO2- alone and in the presence of HgCl2 and ZnCl2 to aged brine at -13 °C resulted in linear increases in the δ15 N of N2 O with time. As HgCl2 and ZnCl2 are effective biocides, we interpret N2 O production in the aged brine to be the result of chemodenitrification. With this understanding, we interpret our results from the field incubations as the result of chemodenitrification stimulated by the addition of 15 N-enriched NO2- and ZnCl2 and determined rates of N2 O and N2 production of 4.11-41.18 and 0.55-1.75 nmol L-1 day-1 , respectively. If these rates are representative of natural production, the current concentration of N2 O in Lake Vida could have been reached between 6 and 465 years. Thus, chemodenitrification alone is sufficient to explain the high levels of N2 O present in Lake Vida.

This study evaluated impacts of the BP Deepwater Horizon ( DWH ) oil spill on petroleum hydrocarbons in surface waters of the Louisiana continental shelf in northern Gulf of Mexico. Surface water (~top 5 cm) without visible oil was collected from three cruises in May 2010 during the oil spill, August 2010 after the well was capped, and May 2011 one year after the spill. Concentrations of total dissolved n -alkanes (C 9 –C 35 ) in surface seawater were more than an order of magnitude higher in May 2010 than August 2010 and May 2011, indicating contamination by the DWH oil spill. This conclusion was further supported by more abundant smaller n -alkanes (C 9 –C 13 ), together with pristane and phytane, in May than August 2010 samples. In contrast, even carbon-numbered dissolved n -alkanes (C 14 –C 20 ) dominated the May 2011 samples, and this distribution pattern of dissolved n -alkanes is the first documentation for water samples in the northern Gulf of Mexico. However, this pattern was not observed in May 2011 suspended particles except for Sta. OSS. This decoupling between dissolved and particle compositions suggests that either these even carbon-numbered n -alkanes originated from bacteria rather than algae, or that the alkanes in the shelf were transported from elsewhere. Concentrations of polycyclic aromatic hydrocarbons (PAHs) in suspended particles were 5 times higher on average in May 2010 (83–252 ng L −1 ) than May 2011 (7.2–83 ng L −1 ), also indicating contamination by the DWH oil spill. Application of a biomarker ratio of 17α(H),21β(H)-30-norhopane over 17α(H),21β(H)-hopane confirmed that suspended particles from at least two stations were contaminated by the DWH oil spill in May 2010. Taken together, these results showed that surface waters of the sampling area in May 2010 were contaminated by the oil spill, but also that rapid weathering and/or physical dilution quickly reduced hydrocarbon levels by August 2010.

Recent global water quality crises point to an urgent need for greater understanding of cyanobacterial harmful algal blooms (cHABs) and their drivers. Nearshore areas of Lake Erie such as Sandusky Bay may become seasonally limited by nitrogen (N) and are characterized by distinct cHAB compositions (i.e., Planktothrix over Microcystis). This study investigated phytoplankton N uptake pathways, determined drivers of N depletion, and characterized the N budget in Sandusky Bay. Nitrate (NO3-) and ammonium (NH4+) uptake, N fixation, and N removal processes were quantified by stable isotopic approaches. Dissimilatory N reduction was a relatively modest N sink, with denitrification, anammox, and N2O production accounting for 84, 14, and 2 % of sediment N removal, respectively. Phytoplankton assimilation was the dominant N uptake mechanism, and NO3- uptake rates were higher than NH4+ uptake rates. Riverine N loading was sometimes insufficient to meet assimilatory and dissimilatory demands, but N fixation alleviated this deficit. N fixation made up 23.7–85.4 % of total phytoplankton N acquisition and indirectly supports Planktothrix blooms. However, N fixation rates were surprisingly uncorrelated with NO3- or NH4+ concentrations. Owing to temporal separation in sources and sinks of N to Lake Erie, Sandusky Bay oscillates between a conduit and a filter of downstream N loading to Lake Erie, delivering extensively recycled forms of N during periods of low export. Drowned river mouths such as Sandusky Bay are mediators of downstream N loading, but climate-change-induced increases in precipitation and N loading will likely intensify N export from these systems.

The continuous rise of atmospheric nitrous oxide (N2O) is an environmental issue of global concern. In biogeochemical studies, N2O production is commonly assumed to arise solely from enzymatic reactions in microbes and fungi. However, iron, manganese and organic compounds readily undergo redox reactions with intermediates in the nitrogen cycle that produce N2O abiotically under relevant environmental conditions at circumneutral pH. Although these abiotic N2O production pathways have been known to occur for close to a century, they are often neglected in modern ecological studies. In this Synthesis and Emerging Ideas paper, we highlight the defining characteristics, environmental controls, and isotopic signatures of abiotic reactions between nitrogen cycle intermediates (hydroxylamine, nitric oxide, and nitrite), redox-active metals (iron and manganese) and organic matter (humic and fulvic acids) that can lead to N2O production. We also discuss the emerging idea that abiotic reactions coupled to biotic processes have widespread ecological relevance and encourage consideration of abiotic production mechanisms in future biogeochemical investigations of N2O cycling.

Recent global water quality crises point to an urgent need for greater understanding of cyanobacterial harmful algal blooms (cHABs) and their drivers. Nearshore areas of Lake Erie such as Sandusky Bay may become seasonally limited by nitrogen (N) and are characterized by distinct cHAB compositions (i.e., Planktothrix over Microcystis). This study investigated phytoplankton N uptake pathways, determined drivers of N depletion, and characterized the N budget in Sandusky Bay. Nitrate (NO3−) and ammonium (NH4+) uptake, N fixation, and N removal processes were quantified by stable isotopic approaches. Dissimilatory N uptake was a relatively modest N sink, with denitrification, anammox, and N2O production accounting for 84, 14, and 2 % of N removal, respectively. Phytoplankton assimilation was the dominant N uptake mechanism, and NO3− uptake rates were higher than NH4+ uptake rates. Riverine DIN loading was sometimes insufficient to meet assimilatory and dissimilatory demands, but N fixation alleviated this deficit. N fixation made up 23.7–85.4 % of total phytoplankton N acquisition and indirectly supports Planktothrix blooms. However, N fixation rates were surprisingly uncorrelated with NO3− or NH4+ concentrations. Owing to temporal separation in sources and sinks of N to Lake Erie, Sandusky Bay pendulums between acting as a strong and weak source of downstream N loading to Lake Erie. Estuarine systems such as Sandusky Bay are mediators of downstream N loading, but climate change-induced increases in precipitation and N loading will likely intensify the swings of the N pendulum in favor of N export.

Lake Vida, located in Victoria Valley, is one of the largest lakes in the McMurdo dry valleys and is known to contain hypersaline liquid brine sealed below 16 m of freshwater ice. For the first time, Lake Vida was drilled to a depth of 27 m. Below 21 m the ice is marked by well-sorted sand layers up to 20 cm thick within a matrix of salty ice. From ice chemistry, isotopic composition of δ18O and δ2H, and ground penetrating radar profiles, we conclude that the entire 27 m of ice formed from surface runoff and the sediment layers represent the accumulation of surface deposits. Radiocarbon and optically stimulated luminescence dating limit the maximum age of the lower ice to 6300 14C yr BP. As the ice cover ablated downwards during periods of low surface inflow, progressive accumulation of sediment layers insulated and preserved the ice and brine beneath, analogous to the processes that preserve shallow ground ice. The repetition of these sediment layers reveals hydrologic variability in Victoria Valley during the mid- to late Holocene. Lake Vida is an exemplar site for understanding the preservation of subsurface brine, ice, and sediment in a cold desert environment.

Soil microbial processes, stimulated by agricultural fertilization, account for 90 % of anthropogenic nitrous oxide (N2O), the leading source of ozone depletion and a potent greenhouse gas. Efforts to reduce N2O flux commonly focus on reducing fertilization rates. Management of microbial processes responsible for N2O production may also be used to reduce N2O emissions, but this requires knowledge of the prevailing process. To this end, stable isotopes of N2O have been applied to differentiate N2O produced by nitrification and denitrification. To better understand the factors contributing to isotopic variation during denitrification, we characterized the δ15N, δ18O and site preference (SP; the intramolecular distribution of 15N in N2O) of N2O produced during NO3− reduction by Pseudomonas chlororaphis subsp. aureofaciens and P. c. subsp. chlororaphis. In addition to species, treatments included electron donor (citrate and succinate) and electron donor concentration (0.01 mM, 0.1 mM, 1 mM, and 10 mM) as factors. In contrast to the expectation of a Rayleigh model, all treatments exhibited curvilinear behaviour between δ15N or δ18O and [−flnf/(1−f)]. The curvilinear behaviour indicates that the fractionation factor changed over the course of the reaction, something that is not unexpected for a multi-step process such as denitrification. Using the derivative of the equation, we estimated that the net isotope effects (η) vary by as much as 100 ‰ over the course of a single reaction, placing challenges for using δ15N and δ18O as apportionment tools. In contrast, SP for denitrification was not affected by the extent of the reaction, the electron donor source, or concentration, although the mean SP of N2O produced by each species differed. Therefore, SP remains a robust indicator of the origin of N2O. To improve apportionment estimates with SP, future studies could evaluate other factors that contribute to the variation in SP.

Molecular nitrogen (N2) comprises three-quarters of Earth's atmosphere and significant portions of other planetary atmospheres. We report a 19 per mil (‰) excess of 15N15N in air relative to a random distribution of nitrogen isotopes, an enrichment that is 10 times larger than what isotopic equilibration in the atmosphere allows. Biological experiments show that the main sources and sinks of N2 yield much smaller proportions of 15N15N in N2. Electrical discharge experiments, however, establish 15N15N excesses of up to +23‰. We argue that 15N15N accumulates in the atmosphere because of gas-phase chemistry in the thermosphere (>100 km altitude) on time scales comparable to those of biological cycling. The atmospheric 15N15N excess therefore reflects a planetary-scale balance of biogeochemical and atmospheric nitrogen chemistry, one that may also exist on other planets.

Upper-atmospheric chemistry and biological N2 cycling engage in a planetary-scale tug-of-war seen in 15N15N of air., Molecular nitrogen (N2) comprises three-quarters of Earth’s atmosphere and significant portions of other planetary atmospheres. We report a 19 per mil (‰) excess of 15N15N in air relative to a random distribution of nitrogen isotopes, an enrichment that is 10 times larger than what isotopic equilibration in the atmosphere allows. Biological experiments show that the main sources and sinks of N2 yield much smaller proportions of 15N15N in N2. Electrical discharge experiments, however, establish 15N15N excesses of up to +23‰. We argue that 15N15N accumulates in the atmosphere because of gas-phase chemistry in the thermosphere (>100 km altitude) on time scales comparable to those of biological cycling. The atmospheric 15N15N excess therefore reflects a planetary-scale balance of biogeochemical and atmospheric nitrogen chemistry, one that may also exist on other planets.

The permanent ice cover of Lake Vida (Antarctica) encapsulates an extreme cryogenic brine ecosystem (−13 °C; salinity, 200). This aphotic ecosystem is anoxic and consists of a slightly acidic (pH 6.2) sodium chloride-dominated brine. Expeditions in 2005 and 2010 were conducted to investigate the biogeochemistry of Lake Vida’s brine system. A phylogenetically diverse and metabolically active Bacteria dominated microbial assemblage was observed in the brine. These bacteria live under very high levels of reduced metals, ammonia, molecular hydrogen (H 2 ), and dissolved organic carbon, as well as high concentrations of oxidized species of nitrogen (i.e., supersaturated nitrous oxide and ∼1 mmol⋅L −1 nitrate) and sulfur (as sulfate). The existence of this system, with active biota, and a suite of reduced as well as oxidized compounds, is unusual given the millennial scale of its isolation from external sources of energy. The geochemistry of the brine suggests that abiotic brine-rock reactions may occur in this system and that the rich sources of dissolved electron acceptors prevent sulfate reduction and methanogenesis from being energetically favorable. The discovery of this ecosystem and the in situ biotic and abiotic processes occurring at low temperature provides a tractable system to study habitability of isolated terrestrial cryoenvironments (e.g., permafrost cryopegs and subglacial ecosystems), and is a potential analog for habitats on other icy worlds where water-rock reactions may cooccur with saline deposits and subsurface oceans.

We used pulsed and continuous additions of 15N together with whole-ecosystem metabolism measurements to elucidate the mechanisms of nitrogen (N) transformations in a small (1170 m2) through-flow wetland situated along a stream. From measurements of the wetland inflow and outflow, we observed a consistent decrease in nitrate (by 10% of inflow concentrations), while ammonium increased by an order of magnitude. Outflow ammonium concentrations oscillated in a diel cycle, inverse to the concentration of dissolved oxygen (i.e., greater ammonium export at night). The pulsed 15N additions showed little uptake of nitrate over time in the wetland and rapid daytime uptake of ammonium from the water column (rate constant, kt 5 0.11 h21). A steady-state 15Nammonium addition demonstrated a similar rate of ammonium uptake (kt 5 0.067 h21), no detectable nitrification, and highlighted the spatial pattern of ammonium and nitrate uptake within the wetland. Porewater concentration profiles suggest high rates of net ammonium diffusion from the sediments. Ecosystem metabolism measurements indicate that release was attenuated during the day by autotrophic uptake, resulting in lower ammonium export during the day. Denitrification rates were modeled from dissolved N2 : Ar ratios, but they were not sufficient to account for the observed loss in nitrate. Nitrate was removed near the pond inflow but not actively cycled throughout the pond, while the balance between sediment release and subsequent uptake of ammonium from the water-column dominated N cycling in this wetland.

Stable isotope addition experiments seeking to trace the denitrification of combined forms of nitrogen (N) to gaseous N2 in aquatic environments typically need to measure the stable isotope ratio of dissolved nitrogen gas. This measurement presents challenges because of the potential for contamination of samples by N in air, and because field experiments conducted in situ often do not produce a marked enrichment in the isotope ratio of the N2 pool. Field experiments also require numerous samples, sometimes processed under arduous conditions, and thus methods have to be convenient and low in cost. This paper describes the methods for sampling and measurement of the N isotope ratio of dissolved nitrogen that were developed for the isotope addition experiments in the Lotic Intersite Nitrogen Experiment (LINX), a cross-site study examining N biogeochemistry in headwater streams. Headspace equilibration was performed in the field and gas samples were stored in re-evacuated glass vials (Exetainers). Samples were processed and stored underwater to minimize the potential for contamination of samples by air. Isotope ratios were measured using a gas chromatograph interfaced to the isotope ratio mass spectrometer and equipped with a custom sample entry system.

Bacteria and functional genes associated with biphenyl (BP) degradation in the root zone of an Austrian pine (Pinus nigra L.) growing naturally in polychlorinated-BP (PCB)-contaminated soil were identified using stable isotope probing (SIP) integrated with comprehensive functional gene analyses. SIP revealed 75 different genera that derived carbon from 13C-BP, with Pseudonocardia, Kribella, Nocardiodes and Sphingomonas predominating carbon acquisition. Rhodococcus spp. were not detected with SIP, despite being the most abundant BP utilizers isolated from agar plates. Only one organism, an Arthrobacter spp., was detected as a BP utilizer by both cultivation and SIP methods. Time-course SIP analyses indicated that secondary carbon flow from BP-utilizing bacteria into other soil organisms may have occurred largely between 4 and 14 days incubation. Functional gene contents of the BP-utilizing metagenome (13C-DNA) were explored using the GeoChip, a functional gene array containing 6465 probes targeting aromatic degradative genes. The GeoChip detected 27 genes, including several associated with catabolism of BP, benzoate and a variety of aromatic ring hydroxylating dioygenase (ARHD) subunits. Genes associated with the beta-ketoadipate pathway were also detected, suggesting a potential role for this plant aromatic catabolic pathway in PCB degradation. Further ARHD analyses using targeted polymerase chain reaction primers and sequence analyses revealed novel dioxygenase sequences in 13C-DNA, including several sequences that clustered distantly from all known ARHDs and others that resembled known Rhodococcus ARHDs. The findings improve our understanding of BP degradation and carbon flow in soil, reveal the extent of culture bias, and may benefit bioremediation research by facilitating the development of molecular tools to detect, quantify and monitor populations involved in degradative processes.

The intramolecular distribution of nitrogen isotopes in N 2 O is an emerging tool for defining the relative importance of microbial sources of this greenhouse gas. The application of intramolecular isotopic distributions to evaluate the origins of N 2 O, however, requires a foundation in laboratory experiments in which individual production pathways can be isolated. Here we evaluate the site preferences of N 2 O produced during hydroxylamine oxidation by ammonia oxidizers and by a methanotroph, ammonia oxidation by a nitrifier, nitrite reduction during nitrifier denitrification, and nitrate and nitrite reduction by denitrifiers. The site preferences produced during hydroxylamine oxidation were 33.5 ± 1.2‰, 32.5 ± 0.6‰, and 35.6 ± 1.4‰ for Nitrosomonas europaea , Nitrosospira multiformis , and Methylosinus trichosporium , respectively, indicating similar site preferences for methane and ammonia oxidizers. The site preference of N 2 O from ammonia oxidation by N. europaea (31.4 ± 4.2‰) was similar to that produced during hydroxylamine oxidation (33.5 ± 1.2‰) and distinct from that produced during nitrifier denitrification by N. multiformis (0.1 ± 1.7‰), indicating that isotopomers differentiate between nitrification and nitrifier denitrification. The site preferences of N 2 O produced during nitrite reduction by the denitrifiers Pseudomonas chlororaphis and Pseudomonas aureofaciens (−0.6 ± 1.9‰ and −0.5 ± 1.9‰, respectively) were similar to those during nitrate reduction (−0.5 ± 1.9‰ and −0.5 ± 0.6‰, respectively), indicating no influence of either substrate on site preference. Site preferences of ∼33‰ and ∼0‰ are characteristic of nitrification and denitrification, respectively, and provide a basis to quantitatively apportion N 2 O.

The natural removal of NO3− by denitrification within riparian zones of streams and rivers is an area of considerable interest owing to its potential to minimize the impacts of excess anthropogenic loadings. In this study we utilize natural variations in stable N isotopic compositions of NO3− and NH4+ within a transect of shallow wells extending 4 m inland from Smith Creek, a southwestern Michigan stream, to provide insight into microbial processes and the extent of NO3− removal within a soil–stream interface. Within this region three water masses with unique biogeochemical characteristics intersect: a shallow flow rich in NH4+ and dissolved organic carbon (DOC), a deep groundwater mass rich in NO3− but depleted in DOC, and stream water low in NO3−, NH4+, and DOC. N isotope values for NO3− within the well transect were highly variable (−7.7–34.1‰) and reflected intense microbial activity within this narrow region. Isotopic variation was primarily controlled by upwelling of deep groundwater near the stream...

The concentration and d 15 No f NO y in leachate from two undisturbed and unfertilized soil lysimeters, one convention- 3

There is much interest in biogeochemical processes that occur at the interface between soils and streams since, at the scale of landscapes, these habitats may function as control points for fluxes of nitrogen (N) and other nutrients from terrestrial to aquatic ecosystems. Here we examine whether a thermodynamic perspective can enhance our mechanistic and predictive understanding of the biogeochemical function of soil–stream interfaces, by considering how microbial communities interact with variations in supplies of electron donors and acceptors. Over a two-year period we analyzed >1400 individual samples of subsurface waters from networks of sample wells in riparian wetlands along Smith Creek, a first-order stream draining a mixed forested–agricultural landscape in southwestern Michigan, USA. We focused on areas where soil water and ground water emerged into the stream, and where we could characterize subsurface flow paths by measures of hydraulic head and/or by in situ additions of hydrologic tracers. We found strong support for the idea that the biogeochemical function of soil–stream interfaces is a predictable outcome of the interaction between microbial communities and supplies of electron donors and acceptors. Variations in key electron donors and acceptors (NO3−, N2O, NH4+, SO42−, CH4, and dissolved organic carbon [DOC]) closely followed predictions from thermodynamic theory. Transformations of N and other elements resulted from the response of microbial communities to two dominant hydrologic flow paths: (1) horizontal flow of shallow subsurface waters with high levels of electron donors (i.e., DOC, CH4, and NH4+), and (2) near-stream vertical upwelling of deep subsurface waters with high levels of energetically favorable electron acceptors (i.e., NO3−, N2O, and SO42−). Our results support the popular notion that soil–stream interfaces can possess strong potential for removing dissolved N by denitrification. Yet in contrast to prevailing ideas, we found that denitrification did not consume all NO3− that reached the soil–stream interface via subsurface flow paths. Analyses of subsurface N chemistry and natural abundances of δ15N in NO3− and NH4+ suggested a narrow near-stream region as functionally the most important location for NO3− consumption by denitrification. This region was characterized by high throughput of terrestrially derived water, by accumulation of dissolved NO3− and N2O, and by low levels of DOC. Field experiments supported our hypothesis that the sustained ability for removal of dissolved NO3− and N2O should be limited by supplies of oxidizable carbon via shallow flowpaths. In situ additions of acetate, succinate, and propionate induced rates of NO3− removal (∼1.8 g N·m−2·d−1) that were orders of magnitude greater than typically reported from riparian habitats. We propose that the immediate near-stream region may be especially important for determining the landscape-level function of many riparian wetlands. Management efforts to optimize the removal of NO3− by denitrification ought to consider promoting natural inputs of oxidizable carbon to this near-stream region.

New radiocarbon age dates for the Cheboygan bryophyte bed in northern lower Michigan indicate that the bed was not deposited during the Mackinaw interstade, as was previously proposed, but is correlative to the Two Creeks forest bed deposited during the Two Creeks interstade approximately 11 850 BP. Furthermore, the till overlying the bryophyte bed does not represent continuous deposition by ice throughout the Two Creeks interstade, as proposed by others, but represents deposition during the Greatlakean stade. A major implication resulting from the reassignment of the age of the Cheboygan bryophyte bed is that the Straits of Mackinac could have been ice free during the Two Creeks interstade and that during that time the Kirkfield phase of glacial Lake Algonquin may have extended into the Lake Michigan basin.

[1] Isotopologue values were used to quantify the contribution of denitrification to nitrous oxide (N2O) flux in agricultural and early successional fields in southwest Michigan. Nitrous oxide-δ15N and δ18O values were poor estimators of microbial origins compared to site preference (SP) (difference in δ15N between the outer and central N atoms of N2O). Site preference was used to evaluate the importance of denitrification (including nitrifier denitrification) in N2O production. Average flux-weighted SP values for each field ranged between 2.9 and 14.6‰ and, on the basis of SP values for N2O production from denitrification (0‰) and collectively for nitrification and fungal denitrification (37‰), these values indicate that between 61 and 92% of N2O originated from denitrification. Reduction of N2O ranged from undetectable to as much as 50% of production; and because reduction increases SP, our results underestimate the percentage of N2O from bacterial denitrification. The SP values in our study clearly indicate that denitrification is the predominant source of soil-derived N2O.

This funding provided support for over nine years of research on the structure and function of microbial communities in subsurface environments. The overarching goal during these years was to understand the impact of mixed contaminants, particularly heavy metals like uranium, on the structure and function of microbial communities. In addition we sought to identify microbial populations that were actively involved in the reduction of metals because these species of bacteria hold the potential for immobilizing soluble metals moving in subsurface water. Bacterial mediated biochemical reduction of metals like uranium, technetium and chromium, greatly reduces their mobility through complexation and precipitation. Hence, by taking advantage of natural metabolic capabilities of subsurface microbial populations it is possible to bioremediate contaminated subsurface environments with a cost-effective in situ approach. Towards this end we have i.) identified bacterial populations that have thrived under the adverse conditions at the contaminated FRC site, ii.) phylogenetically identified populations that respond to imposed remediation conditions at the FRC, iii.) used metagenomics to begin a reconstruction of the metabolic web in a contaminated subsurface zone, iv.) investigated the metal reducing attributes of a Gram-positive spore forming rod also capable of dechlorination.

[1] A study of respiration to photosynthesis (R:P) ratios in Lake Superior, based on the fraction of O2 saturation and the isotopic composition of O2, was undertaken to evaluate spatial and temporal variations in the trophic status of a large oligotrophic freshwater environment. The lake was predominantly net heterotrophic from April to October 2000 (R:P ratios: 1.2–2.5). Uniform R:P ratios of ∼1.5 with depth and across the lake in April 2000 and 2001 revealed the homogeneity of the water column during spring. A brief period of net autotrophy was observed during summer thermal stratification in 2000 and 2001, and surveys showed this condition to be prevalent and lake-wide in August 2001 (R:P ratios: 0.5–0.9). Strong net autotrophy (R:P ratios: 0.6) was found near Duluth, Minnesota, and suggested the potential for the formation of mesotrophic conditions within areas of increased nutrient loadings from urbanization. Respiration and photosynthesis were shown to exert a strong control on O2 gas exchange within Lake Superior, as evidenced by significant correlations between R:P ratios and O2 gas exchange during periods of net heterotrophy and autotrophy. This observation was unexpected since [O2] in the lake appears to be dominated by atmospheric O2 gas exchange, given that the fraction of O2 saturation is continuously near levels expected for equilibration with the atmosphere. Furthermore, the relationship between the biological and physical O2 fluxes may enable the use of R:P ratios to calculate O2 gas exchange and ultimately estimate CO2 fluxes between lakes and the atmosphere.

The relative importance of individual microbial pathways in nitrous oxide (N(2)O) production is not well known. The intramolecular distribution of (15)N in N(2)O provides a basis for distinguishing biological pathways. Concentrated cell suspensions of Methylococcus capsulatus Bath and Nitrosomonas europaea were used to investigate the site preference of N(2)O by microbial processes during nitrification. The average site preference of N(2)O formed during hydroxylamine oxidation by M. capsulatus Bath (5.5 +/- 3.5 per thousand) and N. europaea (-2.3 +/- 1.9 per thousand) and nitrite reduction by N. europaea (-8.3 +/- 3.6 per thousand) differed significantly (ANOVA, f((2,35)) = 247.9, p = 0). These results demonstrate that the mechanisms for hydroxylamine oxidation are distinct in M. capsulatus Bath and N. europaea. The average delta(18)O-N(2)O values of N(2)O formed during hydroxylamine oxidation for M. capsulatus Bath (53.1 +/- 2.9 per thousand) and N. europaea (-23.4 +/- 7.2 per thousand) and nitrite reduction by N. europaea (4.6 +/- 1.4 per thousand) were significantly different (ANOVA, f((2,35)) = 279.98, p = 0). Although the nitrogen isotope value of the substrate, hydroxylamine, was similar in both cultures, the observed fractionation (delta(15)N) associated with N(2)O production via hydroxylamine oxidation by M. capsulatus Bath and N. europaea (-2.3 and 26.0 per thousand, respectively) provided evidence that differences in isotopic fractionation were associated with these two organisms. The site preferences in this study are the first measured values for isolated microbial processes. The differences in site preference are significant and indicate that isotopomers provide a basis for apportioning biological processes producing N(2)O.

There is much interest in biogeochemical processes that occur at the interface between soils and streams since, at the scale of landscapes, these habitats may function as control points for fluxes of nitrogen (N) and other nutrients from terrestrial to aquatic ecosystems. Here we examine whether a thermodynamic perspective can enhance our mechanistic and predictive understanding of the biogeochemical function of soil-stream interfaces, by considering how microbial communities interact with variations in supplies of electron donors and acceptors. Over a two-year period we analyzed >1400 individual samples of subsurface waters from networks of sample wells in riparian wetlands along Smith Creek, a first-order stream draining a mixed forested-agricultural landscape in southwestern Michigan, USA. We focused on areas where soil water and ground water emerged into the stream, and where we could characterize subsurface flow paths by measures of hydraulic head and/or by in situ additions of hydrologic tracers. We found strong support for the idea that the biogeochemical function of soil-stream interfaces is a predictable outcome of the interaction between microbial communities and supplies of electron donors and acceptors. Variations in key electron donors and acceptors (NO 3 - , N 2 O, NH 4 + , SO 4 2- , CH 4 , and dissolved organic carbon [DOC]) closely followed predictions from thermodynamic theory. Transformations of N and other elements resulted from the response of microbial communities to two dominant hydrologic flow paths: (1) horizontal flow of shallow subsurface waters with high levels of electron donors (i.e., DOC, CH 4 , and NH 4 + ), and (2) near-stream vertical upwelling of deep subsurface waters with high levels of energetically favorable electron acceptors (i.e., NO 3 - , N 2 O, and SO 4 2- ). Our results support the popular notion that soil-stream interfaces can possess strong potential for removing dissolved N by denitrification. Yet in contrast to prevailing ideas, we found that denitrification did not consume all NO 3 - that reached the soil-stream interface via subsurface flow paths. Analyses of subsurface N chemistry and natural abundances of δ 15 N in NO 3 - and NH 4 + suggested a narrow near-stream region as functionally the most important location for NO 3 - consumption by denitrification. This region was characterized by high throughput of terrestrially derived water, by accumulation of dissolved NO 3 - and N 2 O, and by low levels of DOC. Field experiments supported our hypothesis that the sustained ability for removal of dissolved NO 3 - and N 2 O should be limited by supplies of oxidizable carbon via shallow flowpaths. In situ additions of acetate, succinate, and propionate induced rates of NO 3 - removal (∼1.8 g N.m -2 .d -1 ) that were orders of magnitude greater than typically reported from riparian habitats. We propose that the immediate near-stream region may be especially important for determining the landscape-level function of many riparian wetlands. Management efforts to optimize the removal of NO 3 - by denitrification ought to consider promoting natural inputs of oxidizable carbon to this near-stream region.