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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.

Atmospheric nitrous oxide (N

© 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.

An understanding of the drivers of hotspot/hot moments of N2O production is required to better constrain the global N2O budget and to plan the mitigation strategies. Hot spots are areas with very high N2O emission rates relative to the surrounding area, while hot moments are short periods of time with very high emission rates. As the decomposition of fresh organic matter is transitory in nature, it may have a strong influence on hotspot and hot moment N2O production. Roots are well known to be hotspots for microbial activity but roots direct contribution to N2O production and emissions in soil remain poorly understood.In this study, we evaluated the role of root decomposition on N2O production and emissions, as a function of soil pore size and water content. We hypothesized that (i) the greatest N2O emissions will be observed from root decomposition in the soil dominated by large (>30 µm Ø) pores due to their high connectivity and (ii) enhanced N2O production by denitrification will be observed due to local anaerobic conditions, generated by O2 consumption by decomposers.To evaluate the role of root decomposition on N2O production we used soil microcosms cultivated with switchgrass (Panicum virgatum L. variety Cave-in-rock). From the same composite soil samples we created two soil materials with contrasting pore architectures, namely soil with prevalence of large pores (≥ 35 μm Ø) and small pores (≤ 10 μm Ø). After four months of growing in a greenhouse, plants were cut and soil microcosms with roots were incubated in the dark at room T for 21 days, at two contrasting soil moisture conditions: 40% and 70% water filled pore space (WFPS). Gas headspace samples were collected at different time points during incubation for N2O and CO2 concentration analysis and isotopic characterization of N2O (δ15Nbulk, site preference (SP), and δ18O).The daily emissions of N2O and CO2 from soil microcosms with grown roots showed the same trend during the incubation period and were significantly higher compared to soil microcosms without roots (control) (p < 0.05). Microcosm with large pores soil had significantly higher N2O flux rates compared to the microcosms with small pore soil for both soil moisture treatments (p < 0.001). The relationship between SP and δ18O (isotope mapping) indicated that heterotrophic bacterial denitrification strongly dominated N2O production between day 1 to 7 of the incubation (≥ 97%) and N2O reduction was higher during this period (40 – 60%) in soil microcosms with both pore size and moisture treatment. Later on, N2O reduction decreased (1 – 35%) while the share of nitrification/fungal sources increased for soil microcosms with large pores.Our results indicated that decomposing roots acted as hotspots enhancing N2O emissions and N2O hotspots occurring during root decomposition are strongly influenced by soil pore architecture. While differences in soil pore architecture did not cause differences in N2O production process at the initial phase of decomposition, it might influence the relative contribution of N2O microbial production pathways in later stage of decomposition.

High temporal and spatial variability of nitrous oxide (N2O) emission from soils has been a challenge for the systematic prediction of global climate change. It is attributed to multiple hotspots occurring simultaneously and affecting the N dynamics cumulatively on an ecosystem scale. Understanding the mechanisms and contributing factors of N2O emission in single hotspots is a prerequisite to overcoming this problem.We investigated the decomposing switchgrass roots as N2O hotspots, using isotope dual-labeling (15N and 13C) and zymography. Our main objectives were i) to quantify the contribution of decomposing roots to N2O emission along with the N contents in the soil (total, organic, and inorganic N) and microbial pools, and ii) to differentiate the extracellular enzyme activity in decomposing roots from the bulk soil, and test if the ‘spatially differentiated’ hotspot enzyme activity indeed related to ‘isotopically differentiated’ hotspot N2O emissions. We treated the soils of the same origin to have different moisture contents (40% and 70% water-filled pore space, WFPS) and pore size distributions (dominant pores of >30 Ø and < 10 mm Ø, referred to as coarse and fine soil), to evaluate how these variables change the contribution of decomposing roots to the N2O production.Our results showed that up to 0.4 % of the root driven N can be emitted as N2O gas, only within 21 days of the decomposition. Approximately 21 ~35% of root N was transformed to dissolved organic N, while less than 1 % of the root N remained as ammonium (NH4+) and nitrate (NO3-) during the incubation. Decreasing NH4+ and increasing NO3- suggested nitrification. Surprisingly, both inorganic and organic N content was greater in coarse soil, which likely led to intense hotspots of enzyme activity and N2O emission. However, there was no difference in microbial biomass between the soil materials. Higher chitinase activity and relatively large pores in coarse soils suggest that the fungal activity was higher in coarse soils compared to the fine soils. Root chitinase activity was positively correlated with the root driven N2O emission rate (p< 0.01, R2=0.22), supporting that the microbial hotspot formed near the root is the hotspots of N2O emission.Our study showed that the intensity of root driven N2O hotspots can highly depend on the soil physical characteristics, being mediated by decomposed substances, and enzyme activity. Tracking the fate of N during the plant root decomposition can provide a new perspective on the strategies to minimize N2O emissions in bioenergy systems.

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.

Restoration of freshwater wetlands presents a potential water quality benefit via removal of nutrients, but complex and unresolved changes in nutrient cycling can occur following restoration. In this study, we evaluated N removal and release in a deltaic wetland under scenarios of hydrologic reconnection and sediment dredging, and we modeled potential downstream impacts of these restoration activities in a Bayesian framework. Denitrification, N2O production, and anammox were measured via the isotope pairing technique in intact sediment cores. Anammox was not detected. Denitrification rates in the control scenario (78.2–87.2 μmol N m−2 h−1) were significantly higher than those under hydrologic reconnection and dredging scenarios (14.7–56.0 μmol N m−2 h−1). N2O production rates were typical of wetland environments. Denitrification and N2O production were stimulated shortly following simulated dredging, indicating a short-term response to sediment disturbance. Under hydrologic reconnection, NH4 + availability was decreased, inhibiting coupled nitrification-denitrification. Despite a decrease in denitrification activity, the wetland has the capacity to remove up to 10% of stream NO3 − following hydrologic reconnection. Restoration is predicted to fully mitigate NH4 + delivery to a downstream eutrophic lake, yet permanent N removal may be reduced due to the decoupling of nitrification and denitrification.

Understanding the magnitude of nitrogen (N) loss and recycling pathways is crucial for coastal N management efforts. However, quantification of denitrification and anammox by a widely-used method, the isotope pairing technique, is challenged when dissimilatory NO3− reduction to NH4+ (DNRA) occurs. In this study, we describe a revised isotope pairing technique that accounts for the influence of DNRA on NO3− reduction (R-IPT-DNRA). The new calculation procedure improves on previous techniques by (1) accounting for N2O production, (2) distinguishing canonical anammox from coupled DNRA-anammox, and (3) including the production of 30N2 by anammox in the quantification of DNRA. This approach avoids the potential for substantial underestimates of anammox rates and overestimates of denitrification rates in systems where DNRA is a significant NO3− reduction pathway. We apply this technique to simultaneously quantify rates of anammox, denitrification, and DNRA in intact sediments adjacent to a seagrass bed in subtropical Australia. The effect of organic carbon lability on NO3− reduction was also addressed by adding detrital sources with differing C:N (phytoplankton- or seagrass-derived). DNRA was the predominant pathway, contributing 49–74% of total NO3− reduction (mean 0.42 µmol N m−2 h−1). In this high C:N system, DNRA outcompetes denitrification for NO3−, functioning to recycle rather than remove N. Anammox exceeded denitrification (mean 0.18 and 0.04 µmol N m−2 h−1, respectively) and accounted for 64–86% of N loss, a rare high percentage in shallow coastal environments. Owing to low denitrification activity, N2O production was ∼100-fold lower than in other coastal sediments (mean 7.7 nmol N m−2 h−1). All NO3− reduction pathways were stimulated by seagrass detritus but not by phytoplankton detritus, suggesting this microbial community is adapted to process organic matter that is typically encountered. The R-IPT-DNRA is widely applicable in other environments where the characterization of co-existing NO3− reduction pathways is desirable.

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.

This study investigated the long-term associations of anthropogenic (sedimentary P, C, and N concentrations, and human population in the watershed), and climatic variables (air temperature, and river discharge) with Escherichia coli uidA and enterococci 23S rRNA concentrations in sediment cores from Anchor Bay (AB) in Lake St. Clair, and near the mouth of the Clinton River (CR), Michigan. Calendar year was estimated from vertical abundances of (137)Cs. The AB and CR cores spanned c.1760-2012 and c.1895-2012, respectively. There were steady state concentrations of enterococci in AB during c.1760-c.1860 and c.1910-c.2003 at ∼0.1 × 10(5) and ∼2.0 × 10(5) cell equivalents (CE) per g-dry wt, respectively. Enterococci concentrations in CR increased toward present day, and ranged from ∼0.03 × 10(5) to 9.9 × 10(5) CE/g-dry wt. The E. coli concentrations in CR and AB increased toward present day, and ranged from 0.14 × 10(7) to 1.7 × 10(7) CE/g-dry wt, and 1.8 × 10(6) to 8.5 × 10(6) CE/g-dry wt, respectively. Enterococci was associated with population and river discharge, while E. coli was associated with population, air temperature, and N and C concentrations (p0.05). Sediments retain records of the abundance of fecal indicator bacteria, and offer a way to evaluate responses to increased population, nutrient loading, and environmental policies.

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.

Increasing seawater temperatures (SWT) resulting from climate change have greatly reduced coral cover world-wide; however, disease has also greatly reduced coral cover in many regions and is especially apparent for offshore bank reefs of the Florida Reef Tract (FRT). The deleterious capacity of future stressors is often dependent upon previous stress. Chronic microbial-related stress has received less attention despite documented links between chronic activation of the innate immune system and the diminished capacity to respond to simultaneously acting stressors. Therefore, differential response of corals to bacterial stress may contribute to decreased coral cover indirectly. To identify differences in bacterial stress response between coral populations, Porites astreoides colonies (n = 6) were collected from an adjacent inshore patch reef and offshore bank-reef of the FRT. Coral were affixed to a concrete substrate at the collection site, and remained there for 2 years to diminish reef environment interspecific effects. Coral were collected after 2 years and subjected to one of three conditions for 8 h: a control condition (28 °C), a thermal stress treatment (32 °C), and thermal and bacterial stress treatment (5 µg mL−1 Serratia marcescens lipopolysaccharide). Offshore reef colonies displayed two- to fourfold increase in innate immune system-related gene expression following temperature stress and LPS, but not with temperature stress alone. Inshore P. astreoides maintained lower levels of expression independent of treatment. These results provide evidence for site-specific sensitivity to bacterial stress and indicate that bacterial stress may indirectly contribute to decreased resistance to climate-related stressors such as SWT.

Rationale Hydrogenases catalyze the reversible formation of H2 from electrons and protons with high efficiency. Understanding the relationships between H2 production, H2 uptake, and H2-H2O exchange can provide insight into the metabolism of microbial communities in which H2 is an essential component in energy cycling. Methods We used stable H isotopes (1H and 2H) to probe the isotope effects associated with three [FeFe]-hydrogenases and three [NiFe]-hydrogenases. Results All six hydrogenases displayed fractionation factors for H2 formation that were significantly less than 1, producing H2 that was severely depleted in 2H relative to the substrate, water. Consistent with differences in their active site structure, the fractionation factors for each class appear to cluster, with the three [NiFe]-hydrogenases (α = 0.27–0.40) generally having smaller values than the three [FeFe]-hydrogenases (α = 0.41–0.55). We also obtained isotopic fractionation factors associated with H2 uptake and H2-H2O exchange under conditions similar to those utilized for H2 production, providing a more complete picture of the reactions catalyzed by hydrogenases. Conclusions The fractionation factors determined in our studies can be used as signatures for different hydrogenases to probe their activity under different growth conditions and to ascertain which hydrogenases are most responsible for H2 production and/or uptake in complex microbial communities. Copyright © 2015 John Wiley & Sons, Ltd.

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.

We evaluated O 2 metabolism and mixing of river and seawater in the northern Gulf of Mexico (NGOMEX) in May and August of 2010. A strong correlation between the δ 2 H and δ 18 O of water and salinity indicated simple mixing of the two water sources. The isotope values for the freshwater source suggested that Mississippi River water predominated over the Atchafalaya River water in at our study sites in both May and August. Knowledge of the δ 18 O of water is required for determination of the ratio of primary production to respiration ( P : R ) from the δ 18 O and concentration of O 2 . We observed a strong correlation between δ 18 O of water and salinity, which indicates that subsequent studies of P : R may simply be able to predict the isotopic composition of water from salinity in this region. P : R values were determined from the concentration and isotopic composition of O 2 and varied from 0.88 to 1.10. The range in P : R and progression from high values in May to net heterotrophy in August is consistent with enhanced primary production in spring followed by respiration in excess of production in summer. Averages of −5.8‰ and −3.4‰ were observed for the isotopic enrichment factor, e obs , for sub-pycnocline respiration in May and August, respectively. In contrast to previous studies we found that e obs is not an effective indicator of the proportion of water column to sediment respiration, but rather reflects the total rate of sub-pycnocline respiration (water column plus sediment respiration). Nonetheless, as e obs is an indication of total sub-pycnocline respiration insight into oxygen-removal can be obtained without the need for incubation. Even though our research cruises occurred approximately three weeks after the initiation and one month after cessation of the Deepwater Horizon oil spill, P : R and e obs values were each within ranges reported previously in NGOMEX. Thus, a direct link between the oil spill and O 2 metabolism or hypoxia development was not apparent.

RATIONALE In recent years, research and applications of the N2O site-specific nitrogen isotope composition have advanced, reflecting awareness of the contribution of N2O to the anthropogenic greenhouse effect, and leading to significant progress in instrument development. Further dissemination of N2O isotopomer analysis, however, is hampered by a lack of internationally agreed gaseous N2O reference materials and an uncertain compatibility of different laboratories and analytical techniques. METHODS In a first comparison approach, eleven laboratories were each provided with N2O at tropospheric mole fractions (target gas T) and two reference gases (REF1 and REF2). The laboratories analysed all gases, applying their specific analytical routines. Compatibility of laboratories was assessed based on N2O isotopocule data for T, REF1 and REF2. Results for T were then standardised using REF1 and REF2 to evaluate the potential of N2O reference materials for improving compatibility between laboratories. RESULTS Compatibility between laboratories depended on the analytical technique: isotope ratio mass spectrometry (IRMS) results showed better compatibility for δ(15)N values, while the performance of laser spectroscopy was superior with respect to N2O site preference. This comparison, however, is restricted by the small number of participating laboratories applying laser spectroscopy. Offset and two-point calibration correction of the N2O isotopomer data significantly improved the consistency of position-dependent nitrogen isotope data while the effect on δ(15)N values was only minor. CONCLUSIONS The study reveals that for future research on N2O isotopocules, standardisation against N2O reference material is essential to improve interlaboratory compatibility. For atmospheric monitoring activities, we suggest N2O in whole air as a unifying scale anchor.

Hydrogenases catalyze the reversible formation of H2 from electrons and protons with high efficiency. Understanding the relationships between H2 production, H2 uptake, and H2-H2O exchange can provide insight into the metabolism of microbial communities in which H2 is an essential component in energy cycling.We used stable H isotopes (1H and 2H) to probe the isotope effects associated with three [FeFe]-hydrogenases and three [NiFe]-hydrogenases.All six hydrogenases displayed fractionation factors for H2 formation that were significantly less than 1, producing H2 that was severely depleted in 2H relative to the substrate, water. Consistent with differences in their active site structure, the fractionation factors for each class appear to cluster, with the three [NiFe]-hydrogenases (α = 0.27–0.40) generally having smaller values than the three [FeFe]-hydrogenases (α = 0.41–0.55). We also obtained isotopic fractionation factors associated with H2 uptake and H2-H2O exchange under conditions similar to those utilized for H2 production, providing a more complete picture of the reactions catalyzed by hydrogenases.The fractionation factors determined in our studies can be used as signatures for different hydrogenases to probe their activity under different growth conditions and to ascertain which hydrogenases are most responsible for H2 production and/or uptake in complex microbial communities.

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.

Summary 1. Nitrogen (N) processing in streams has been investigated using whole-stream 15N addition experiments that, in general, have found that a large proportion of added nitrate removed from the water column appears to be assimilated by the stream benthos. The long-term fate of this retained N is unknown, and of particular interest is the possibility that it becomes denitrified through coupled mineralisation–nitrification–denitrification processes (indirect denitrification). 2. We used in situ chambers to produce highly 15N-enriched benthic biofilms and removed the chambers to allow biofilms to interact with ambient stream conditions. Nitrogen assimilation and direct denitrification were estimated from the first chamber deployment. Chambers were periodically reinstalled over 4 weeks to measure tracer 15N in ammonium (), nitrate () and dinitrogen (N2), from which we estimated subsequent rates of biotic N transformations, including N mineralisation (ammonification), nitrification and indirect denitrification. We also estimated rates of depuration of 15N tracer from benthic biomass compartments. 3. Nitrate uptake was roughly equivalent in the sand and cobble habitats that dominated the stream. Direct denitrification (denitrification of from the water column) was an order of magnitude higher in cobble habitats than in sand habitats, accounting for c. 26 and 2% of total nitrate uptake in cobble and sand, respectively. 4. Mean residence times of actively cycling organic N in stream benthos (algae and microbes) were 16 days in cobble habitats and 9 days in sand habitats. The difference between habitat types was driven by the influence of N residence time in epilithic biofilms (18 days) on cobbles. 5. Release of enriched 15 was the primary flux of remineralised N, while release of enriched 15 was an order of magnitude less. We detected slight 15N enrichment in dissolved nitrogen gas (N2) in post-enrichment sampling, indicating that indirect denitrification was taking place. However, indirect denitrification accounted for

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.

Hydrogenases catalyze the reversible formation of H(2), and they are key enzymes in the biological cycling of H(2). H isotopes have the potential to be a very useful tool in quantifying hydrogen ion trafficking in biological H(2) production processes, but there are several obstacles that have thus far limited the application of this tool. Here, we describe a new method that overcomes some of these barriers and is specifically designed to measure isotopic fractionation during enzyme-catalyzed H(2) evolution. A key feature of this technique is that purified hydrogenases are employed, allowing precise control over the reaction conditions and therefore a high level of precision. In addition, a custom-designed high-throughput gas chromatograph/isotope ratio mass spectrometer is employed to measure the isotope ratio of the H(2). Using our new approach, we determined that the fractionation factor for H(2) production by the [NiFe]-hydrogenase from Desulfovibrio fructosovorans is 0.273 ± 0.006. This result indicates that, as expected, protons are highly favored over deuterium ions during H(2) evolution. Potential applications of this newly developed method are discussed.

Nitrous oxide (N2O) is an important greenhouse gas that is emitted from soil, but obtain- ing precise N2O source and sink strength estimates has been difficult due to high spatial and temporal flux variability and a poor understanding of the mechanisms controlling fluxes. Tools that improve our ability to quantify trace gas fluxes from soil and constrain annual budgets are therefore needed. Here we describe an improved chamber-based sampling system that continuously traps evolving soil gases onto molecular sieve thereby obtaining a single sample that integrates fluxes over extended periods (several weeks or more) and the use of stable isotopic methods to study microbial origins of N2O. We demonstrate that N2O can be trapped on molecular sieve within our chamber system with near 100% recovery and without isotopic fractionation. In field trials the site preference of N2O (the difference in d 15 N between the central and outer N atoms) varied between -6 and 14.4%, indicating that the majority of flux was derived from bacterial denitrification. Further development with automation would improve flux estimates by providing a system capable of capturing episodic flux events owing to long-term deployment. Further, an automated trapping chamber approach will also provide process-based understand- ing of N2O dynamics via stable isotopes and a new and affordable tool for evaluating the response of trace gas fluxes to land management practices.

The source of N 2 O in terrestrial ecosystems has long been debated. Both nitrification and denitrification produce N 2 O but their relative importance remains uncertain. Here we apply site preference, SP (the difference in δ 15 N between the central and outer N atom in N 2 O), to estimate the relative importance of bacterial denitrification (including nitrifier denitrification) to total N 2 O production from soil. We measured SP over a diurnal cycle following the third year of tillage of a previously uncultivated grassland soil at the Kellogg Biological Station (KBS) in southwestern Michigan. Fluxes of N 2 O in our study ranged between 7.8 and 12.1 g N 2 O–N ha −1 d −1 and were approximately 3 and 10 times greater than fluxes observed in managed agricultural and successional fields, respectively, at KBS. Consequently, our study captured a period of high flux resulting from the cultivation of a historically never-tilled soil. Concentration weighted SP values decreased from 12.9‰ in the morning to a minimum value of −0.1‰ in the afternoon. Based on SP values reported for bacterial denitrification (−5 to 0‰; Toyoda et al., 2005; Sutka et al., 2006 ), hydroxylamine oxidation (nitrification) and fungal denitrification (33–37‰; Sutka et al., 2006 ) we found that production attributable to bacterial denitrification increased from between 52.9 and 60.9% in the morning to between 87.5 and 100% in the afternoon. Further, we observed diurnal variation in flux and SP that is consistent with increased production from bacterial denitrification associated with temperature-driven increases in respiration.

Identifying the importance of fungi to nitrous oxide (N2O) production requires a non-intrusive method for differentiating between fungal and bacterial N2O production such as natural abundance stable isotopes. We compare the isotopologue composition of N2O produced during nitrite reduction by the fungal denitrifiers Fusarium oxysporum and Cylindrocarpon tonkinense with published data for N2O production during bacterial nitrification and denitrification. The fractionation factors for bulk nitrogen isotope values for fungal denitrification were in the range −74.7 to −6.6‰. There was an inverse relationship between the absolute value of the fractionation factors and the reaction rate constant. We interpret this in terms of variation in the relative importance of the rate constants for diffusion and enzymatic reduction in controlling the net isotope effect for N2O production during fungal denitrification. Over the course of nitrite reduction, the δ18O values for N2O remained constant and did not exhibit a relationship with the concentration characteristic of an isotope effect. This probably reflects isotopic exchange with water. Similar to the δ18O data, the site preference (SP; the difference in δ15N between the central and outer N atoms in N2O) was unrelated to concentration during nitrite reduction and, therefore, has the potential to act as a conservative tracer of production from fungal denitrification. The SP values of N2O produced by F. oxysporum and C. tonkinense were 37.1 ± 2.5‰ and 36.9 ± 2.8‰, respectively. These SP values are similar to those obtained in pure culture studies of bacterial nitrification but quite distinct from SP values for bacterial denitrification. The large magnitude of the bulk nitrogen isotope fractionation and the δ18O values associated with fungal denitrification are distinct from bacterial production pathways; thus multiple isotopologue data holds much promise for resolving bacterial and fungal production. Our work further provides insight into the role that fungal and bacterial nitric oxide reductases have in determining site preference during N2O production. Copyright © 2008 John Wiley & Sons, Ltd.

Isotopologue analyses of N2O within soil mesocosm experiments were used to evaluate the influence of N2O reduction on isotope fractionation. We investigated fractionation during N2O reduction at 60%, 80% and 100% water-filled pore space (WFPS) and found net isotope effects (NIE) for δ15N of 4.2–7.8‰, δ18O of 12.5–19.1‰, δ15Nα of 6.4–9.7‰ and δ15Nβ of 2.0–5.9‰. Consequently, N2O reduction has a marked affect on isotopologue values and the importance of this process in flux chamber studies should not be ignored. With the exception of SP (the difference between the δ15N of the central, α, and terminal, β, atoms) inverse relationships between the NIE, reaction rate and reaction rate constant and WFPS were observed. Isotopic discrimination in SP during N2O reduction was small and the average NIE for the treatments varied between 2.9‰ and 4.5‰. A strong correlation was evident between δ18O vs. δ15N and δ18O vs. δ15Nα during reduction with slopes of 2.6 and 1.9, respectively, which contrasts from a slope of

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.