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The sources and sinks of nitrous oxide, as control emissions to the atmosphere, are generally poorly constrained for most environmental systems. Initial depth-resolved analysis of nitrous oxide flux from observation wells and the proximal surface within a nitrate contaminated aquifer system revealed high subsurface production but little escape from the surface. To better understand the environmental controls of production and emission at this site, we used a combination of isotopic, geochemical, and molecular analyses to show that chemodenitrification and bacterial denitrification are major sources of nitrous oxide in this subsurface, where low DO, low pH, and high nitrate are correlated with significant nitrous oxide production. Depth-resolved metagenomes showed that consumption of nitrous oxide near the surface was correlated with an enrichment of Clade II nitrous oxide reducers, consistent with a growing appreciation of their importance in controlling release of nitrous oxide to the atmosphere. Our work also provides evidence for the reduction of nitrous oxide at a pH of 4, well below the generally accepted limit of pH 5.

Microbially mediated cycling processes play central roles in regulating the speciation and availability of nitrogen, a vital nutrient with wide implications for agriculture, water quality, wastewater treatment, ecosystem health, and climate change. Ammonia oxidation, the first and rate-limiting step of nitrification, is carried out by bacteria (AOB) and archaea (AOA) that require the trace metal micronutrients copper (Cu) and iron (Fe) for growth and metabolic catalysis. While stable isotope analyses for constraining nitrogen cycling are commonly used, it is unclear whether metal availability may modulate expression of stable isotope fractionation during ammonia oxidation, by varying growth or through regulation of metabolic metalloenzymes. We present the first study examining the influence of Fe and Cu availability on the kinetic nitrogen isotope effect in ammonia oxidation ( 15 ε AO ). We report a general independence of 15 ε AO from the growth rate in AOB, except at a low temperature (10 °C). With AOA Nitrosopumilus maritimus SCM1, however, 15 ε AO decreases nonlinearly at lower oxidation rates. We examine assumptions involved in the interpretation of 15 ε AO values and suggest these dynamics may arise from physiological constraints that push the system toward isotopic equilibrium. These results suggest important links between isotope fractionation and environmental constraints on physiology in these key N cycling microorganisms.

Reactive oxygen species (ROS) are central to diverse biological processes through which organisms respond to and interact with their surroundings. Yet, a lack of direct measurements limits our understanding of the distribution of ROS in the ocean. Using a recently developed in situ sensor, we show that deep-sea corals and sponges produce the ROS superoxide, revealing that benthic organisms can be sources and hotspots of ROS production in these environments. These findings confirm previous contentions that extracellular superoxide production by corals can be independent of the activity of photosynthetic symbionts. The discovery of deep-sea corals and sponges as sources of ROS has implications for the physiology and ecology of benthic organisms and introduces a previously overlooked suite of redox reactants at depth., (© The Author(s) 2023. Published by Oxford University Press on behalf of National Academy of Sciences.)

Around 50% of humankind relies on groundwater as a source of drinking water. Here we investigate the age, geochemistry, and microbiology of 138 groundwater samples from 95 monitoring wells (<250 m depth) located in 14 aquifers in Canada. The geochemistry and microbiology show consistent trends suggesting large-scale aerobic and anaerobic hydrogen, methane, nitrogen, and sulfur cycling carried out by diverse microbial communities. Older groundwaters, especially in aquifers with organic carbon-rich strata, contain on average more cells (up to 1.4 × 10 7  mL -1 ) than younger groundwaters, challenging current estimates of subsurface cell abundances. We observe substantial concentrations of dissolved oxygen (0.52 ± 0.12 mg L -1 [mean ± SE]; n = 57) in older groundwaters that seem to support aerobic metabolisms in subsurface ecosystems at an unprecedented scale. Metagenomics, oxygen isotope analyses and mixing models indicate that dark oxygen is produced in situ via microbial dismutation. We show that ancient groundwaters sustain productive communities and highlight an overlooked oxygen source in present and past subsurface ecosystems of Earth., (© 2023. The Author(s).)

The biogeochemical fluxes that cycle oxygen (O 2 ) play a critical role in regulating Earth's climate and habitability. Triple-oxygen isotope (TOI) compositions of marine dissolved O 2 are considered a robust tool for tracing oxygen cycling and quantifying gross photosynthetic O 2 production. This method assumes that photosynthesis, microbial respiration, and gas exchange with the atmosphere are the primary influences on dissolved O 2 content, and that they have predictable, fixed isotope effects. Despite its widespread use, there are major elements of this approach that remain uncharacterized, including the TOI dynamics of respiration by marine heterotrophic bacteria and abiotic O 2 sinks such as the photochemical oxidation of dissolved organic carbon (DOC). Here, we report the TOI fractionation for O 2 utilization by two model marine heterotrophs and by abiotic photo-oxidation of representative terrestrial and coastal marine DOC. We demonstrate that TOI slopes associated with these processes span a significant range of the mass-dependent domain (λ = 0.499 to 0.521). A sensitivity analysis reveals that even under moderate productivity and photo-oxidation scenarios, true gross oxygen production may deviate from previous estimates by more than 20% in either direction. By considering a broader suite of oxygen cycle reactions, our findings challenge current gross oxygen production estimates and highlight several paths forward to better understanding the marine oxygen and carbon cycles., (© The Author(s) 2022. Published by Oxford University Press on behalf of the National Academy of Sciences.)

Reactive oxygen species (ROS) are key drivers of biogeochemical cycling while also exhibiting both positive and negative effects on marine ecosystem health. However, quantification of the ROS superoxide (O 2 - ) within environmental systems is hindered by its short half-life. Recently, the development of the diver-operated submersible chemiluminescent sensor (DISCO), a submersible, handheld instrument, enabled in situ superoxide measurements in real time within shallow coral reef ecosystems. Here, we present a redesigned and improved instrument, DISCO II. Similar to the previous DISCO, DISCO II is a self-contained, submersible sensor, deployable to 30 m depth and capable of measuring reactive intermediate species in real time. DISCO II is smaller, lighter, lower cost, and more robust than its predecessor. Laboratory validation of DISCO II demonstrated an average limit of detection in natural seawater of 133.1 pM and a percent variance of 0.7%, with stable photo multiplier tube (PMT) counts, internal temperature, and flow rates. DISCO II can also be optimized for diverse environmental conditions by adjustment of the PMT supply voltage and integration time. Field tests showed no drift in the data with a percent variance of 3.0%. Wand tip adaptations allow for in situ calibrations and decay rates of superoxide using a chemical source of superoxide (SOTS-1). Overall, DISCO II is a versatile, user-friendly sensor that enables measurements in diverse environments, thereby improving our understanding of the cycling of reactive intermediates, such as ROS, across various marine ecosystems.

Natural-abundance measurements of nitrate and nitrite (NO x ) isotope ratios (δ 15 N and δ 18 O) can be a valuable tool to study the biogeochemical fate of NO x species in the environment. A prerequisite for using NO x isotopes in this regard is an understanding of the mechanistic details of isotope fractionation ( 15 ε, 18 ε) associated with the biotic and abiotic NO x transformation processes involved (e.g., denitrification). However, possible impacts on isotope fractionation resulting from changing growth conditions during denitrification, different carbon substrates, or simply the presence of compounds that may be involved in NO x reduction as co-substrates [e.g., Fe(II)] remain uncertain. Here we investigated whether the type of organic substrate, i.e., short-chained organic acids, and the presence/absence of Fe(II) (mixotrophic vs. heterotrophic growth conditions) affect N and O isotope fractionation dynamics during nitrate (NO 3 - ) and nitrite (NO 2 - ) reduction in laboratory experiments with three strains of putative nitrate-dependent Fe(II)-oxidizing bacteria and one canonical denitrifier. Our results revealed that 15 ε and 18 ε values obtained for heterotrophic ( 15 ε-NO 3 - : 17.6 ± 2.8‰, 18 ε-NO 3 - :18.1 ± 2.5‰; 15 ε-NO 2 - : 14.4 ± 3.2‰) vs. mixotrophic ( 15 ε-NO 3 - : 20.2 ± 1.4‰, 18 ε-NO 3 - : 19.5 ± 1.5‰; 15 ε-NO 2 - : 16.1 ± 1.4‰) growth conditions are very similar and fall within the range previously reported for classical heterotrophic denitrification. Moreover, availability of different short-chain organic acids (succinate vs. acetate), while slightly affecting the NO x reduction dynamics, did not produce distinct differences in N and O isotope effects. N isotope fractionation in abiotic controls, although exhibiting fluctuating results, even expressed transient inverse isotope dynamics ( 15 ε-NO 2 - : -12.4 ± 1.3 ‰). These findings imply that neither the mechanisms ordaining cellular uptake of short-chain organic acids nor the presence of Fe(II) seem to systematically impact the overall N and O isotope effect during NO x reduction. The similar isotope effects detected during mixotrophic and heterotrophic NO x reduction, as well as the results obtained from the abiotic controls, may not only imply that the enzymatic control of NO x reduction in putative NDFeOx bacteria is decoupled from Fe(II) oxidation, but also that Fe(II) oxidation is indirectly driven by biologically (i.e., via organic compounds) or abiotically (catalysis via reactive surfaces) mediated processes co-occurring during heterotrophic denitrification., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Visser, Wankel, Frey, Kappler and Lehmann.)

Based on knowledge of their production pathways, and limited discrete observations, a variety of short-lived chemical species are inferred to play active roles in chemical cycling in the sea. In some cases, these species may exert a disproportionate impact on marine biogeochemical cycles, affecting the redox state of metal and carbon, and influencing the interaction between organisms and their environment. One such short-lived chemical is superoxide, a reactive oxygen species (ROS), which undergoes a wide range of environmentally important reactions. Yet, due to its fleeting existence which precludes traditional shipboard analyses, superoxide concentrations have never been characterized in the deep sea. To this end, we have developed a submersible oceanic chemiluminescent analyzer of reactive intermediate species (SOLARIS) to enable continuous measurements of superoxide at depth. Fluidic pumps on SOLARIS combine seawater for analysis with reagents in a spiral mixing cell, initiating a chemiluminescent reaction that is monitored by a photomultiplier tube. The superoxide in seawater is then related to the quantity of light produced. Initial field deployments of SOLARIS have revealed high-resolution trends in superoxide throughout the water column. SOLARIS presents the opportunity to constrain the distributions of superoxide, and any number of chemiluminescent species in previously unexplored environments.

Oceanic oxygen minimum zones (OMZs) are globally significant sites of biogeochemical cycling where microorganisms deplete dissolved oxygen (DO) to concentrations <20 µM. Amid intense competition for DO in these metabolically challenging environments, aerobic nitrite oxidation may consume significant amounts of DO and help maintain low DO concentrations, but this remains unquantified. Using parallel measurements of oxygen consumption rates and 15 N-nitrite oxidation rates applied to both water column profiles and oxygen manipulation experiments, we show that the contribution of nitrite oxidation to overall DO consumption systematically increases as DO declines below 2 µM. Nitrite oxidation can account for all DO consumption only under DO concentrations <393 nM found in and below the secondary chlorophyll maximum. These patterns are consistent across sampling stations and experiments, reflecting coupling between nitrate reduction and nitrite-oxidizing Nitrospina with high oxygen affinity (based on isotopic and omic data). Collectively our results demonstrate that nitrite oxidation plays a pivotal role in the maintenance and biogeochemical dynamics of OMZs., (© 2021. The Author(s).)

Oceanic deoxygenation is increasingly affecting marine ecosystems; many taxa will be severely challenged, yet certain nominally aerobic foraminifera (rhizarian protists) thrive in oxygen-depleted to anoxic, sometimes sulfidic, sediments uninhabitable to most eukaryotes. Gene expression analyses of foraminifera common to severely hypoxic or anoxic sediments identified metabolic strategies used by this abundant taxon. In field-collected and laboratory-incubated samples, foraminifera expressed denitrification genes regardless of oxygen regime with a putative nitric oxide dismutase, a characteristic enzyme of oxygenic denitrification. A pyruvate:ferredoxin oxidoreductase was highly expressed, indicating the capability for anaerobic energy generation during exposure to hypoxia and anoxia. Near-complete expression of a diatom's plastid genome in one foraminiferal species suggests kleptoplasty or sequestration of functional plastids, conferring a metabolic advantage despite the host living far below the euphotic zone. Through a unique integration of functions largely unrecognized among "typical" eukaryotes, benthic foraminifera represent winning microeukaryotes in the face of ongoing oceanic deoxygenation., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

Coral reef health depends on an intricate relationship among the coral animal, photosynthetic algae, and a complex microbial community. The holobiont can impact the nutrient balance of their hosts amid an otherwise oligotrophic environment, including by cycling physiologically important nitrogen compounds. Here we use 15 N-tracer experiments to produce the first simultaneous measurements of ammonium oxidation, nitrate reduction, and nitrous oxide (N 2 O) production among five iconic species of reef-building corals (Acropora palmata, Diploria labyrinthiformis, Orbicella faveolata, Porites astreoides, and Porites porites) in the highly protected Jardines de la Reina reefs of Cuba. Nitrate reduction is present in most species, but ammonium oxidation is low potentially due to photoinhibition and assimilatory competition. Coral-associated rates of N 2 O production indicate a widespread potential for denitrification, especially among D. labyrinthiformis, at rates of ~1 nmol cm -2  d -1 . In contrast, A. palmata displays minimal active nitrogen metabolism. Enhanced rates of nitrate reduction and N 2 O production are observed coincident with dark net respiration periods. Genomes of bacterial cultures isolated from multiple coral species confirm that microorganisms with the ability to respire nitrate anaerobically to either dinitrogen gas or ammonium exist within the holobiont. This confirmation of anaerobic nitrogen metabolisms by coral-associated microorganisms sheds new light on coral and reef productivity.

Rationale: Oxygen isotope ratio measurements of NO 2 - and NO 3 - by the azide method and denitrifier method are sensitive to the δ 18 O value of the sample water. However, the influence of δ 18 O H2O on those measurements has not been quantitatively evaluated and documented so far. Therefore, we investigated the influence of δ 18 O H2O of a sample on the δ 18 O analysis of NO 2 - and NO 3 - ., Methods: We prepared NO 2 - and NO 3 - standards (with known δ 18 O NO2- and δ 18 O NO3- values) dissolved in waters having different δ 18 O H2O values (δ 18 O H2O  = -12.6, 25.9, 56.7, and 110.1‰). Nitrite and nitrate were converted into N 2 O using the azide method and the denitrifier method, respectively. The isotope ratios of the generated N 2 O were measured with a Sercon purge-and-trap gas chromatography/isotope ratio mass spectrometry (PT-GC/IRMS) system. The measured δ 18 O values of the produced N 2 O were plotted against known δ 18 O NO2- and δ 18 O NO3- values to evaluate the influence of exchange of an oxygen atom with H 2 O during the conversion of NO 2 - into N 2 O and NO 3 - into N 2 O, respectively., Results: The degree of oxygen isotope exchange was 10.8 ± 0.3% in the azide method and 5.5 ± 1.0% in the denitrifier method, indicating that the azide method is more susceptible to artifacts arising from differences in the δ 18 O H2O value of water than the denitrifier method. Thus, the intercept of the standard calibration curve must be corrected to account for differences in δ 18 O H2O . Abiotic NO 2 -H 2 O equilibrium isotope effect experiments yielded a rate constant of (1.13 ± 007) × 10 -2 (h -1 ) and an equilibrium isotope effect of 11.9 ± 0.1‰ under the condition of pH = 7.5, 30°C, and 2.5% salinity., Conclusions: Oxygen isotope ratio measurements of NO 2 - by the azide method are highly sensitive to δ 18 O H2O as a result of significant oxygen isotope exchange between NO 2 - and H 2 O. Therefore, to obtain the most accurate measurements water with the same δ 18 O H2O value as that of the sample must be used to make the NO 2 - and NO 3 - standards., (© 2020 John Wiley & Sons, Ltd.)

In the marine environment, the reactive oxygen species (ROS) superoxide is produced through a diverse array of light-dependent and light-independent reactions, the latter of which is thought to be primarily controlled by microorganisms. Marine superoxide production influences organic matter remineralization, metal redox cycling, and dissolved oxygen concentrations, yet the relative contributions of different sources to total superoxide production remain poorly constrained. Here we investigate the production, steady-state concentration, and particle-associated nature of light-independent superoxide in productive waters off the northeast coast of North America. We find exceptionally high levels of light-independent superoxide in the marine water column, with concentrations ranging from 10 pM to in excess of 2,000 pM. The highest superoxide concentrations were particle associated in surface seawater and in aphotic seawater collected meters off the seafloor. Filtration of seawater overlying the continental shelf lowered the light-independent, steady-state superoxide concentration by an average of 84%. We identify eukaryotic phytoplankton as the dominant particle-associated source of superoxide to these coastal waters. We contrast these measurements with those collected at an off-shelf station, where superoxide concentrations did not exceed 100 pM, and particles account for an average of 40% of the steady-state superoxide concentration. This study demonstrates the primary role of particles in the production of superoxide in seawater overlying the continental shelf and highlights the importance of light-independent, dissolved-phase reactions in marine ROS production., (©2020. The Authors.)

The Rhône River originates in the high Alps and drains an intensely cultivated and industrialised catchment before it discharges to the Gulf of Lion. We investigated the interaction of catchment geomorphology with nitrate sources (atmosphere, agriculture, and nitrification of soil organic matter) and removal processes in large and diverse watersheds on the basis of dual nitrate isotope signatures in river water.In March 2015, we took surface water samples along the Rhône River, including its main tributaries, and measured nutrient concentrations and the stable isotopic composition of nitrate (δ 15 N, δ 18 O and Δ 17 O), and water (δ 18 O-H 2 O).Results show that high altitude regions are dominated by nitrate from nitrification in pristine soils and atmospheric deposition, while nitrate in the downstream Rhône River originates mainly from nitrification of agricultural/urban sources. Parallel increases in δ 15 N and δ 18 O reflect the influence of primary production. Previous studies suggested robust correlations between land use and [Formula: see text]. Based on our observation that nitrate δ 15 N values at higher altitudes are lower than expected, we assume that lower nitrate δ 15 N values likely reflect limited nitrate consumption and lower soil nitrogen turnover rates. We propose that correlation between land use and nitrate δ 15 N is sensitive to slope and geomorphology.

The balance between sources and sinks of molecular oxygen in the oceans has greatly impacted the composition of Earth's atmosphere since the evolution of oxygenic photosynthesis, thereby exerting key influence on Earth's climate and the redox state of (sub)surface Earth. The canonical source and sink terms of the marine oxygen budget include photosynthesis, respiration, photorespiration, the Mehler reaction, and other smaller terms. However, recent advances in understanding cryptic oxygen cycling, namely the ubiquitous one-electron reduction of O 2 to superoxide by microorganisms outside the cell, remains unexplored as a potential player in global oxygen dynamics. Here we show that dark extracellular superoxide production by marine microbes represents a previously unconsidered global oxygen flux and sink comparable in magnitude to other key terms. We estimate that extracellular superoxide production represents a gross oxygen sink comprising about a third of marine gross oxygen production, and a net oxygen sink amounting to 15 to 50% of that. We further demonstrate that this total marine dark extracellular superoxide flux is consistent with concentrations of superoxide in marine environments. These findings underscore prolific marine sources of reactive oxygen species and a complex and dynamic oxygen cycle in which oxygen consumption and corresponding carbon oxidation are not necessarily confined to cell membranes or exclusively related to respiration. This revised model of the marine oxygen cycle will ultimately allow for greater reconciliation among estimates of primary production and respiration and a greater mechanistic understanding of redox cycling in the ocean., Competing Interests: The authors declare no competing interest., (Copyright © 2020 the Author(s). Published by PNAS.)

Gas ebullition from aquatic systems to the atmosphere represents a potentially important fraction of primary production that goes unquantified by measurements of dissolved gas concentrations. Although gas ebullition from photosynthetic surfaces has often been observed, it is rarely quantified. The resulting underestimation of photosynthetic activity may significantly bias the determination of ecosystem trophic status and estimated rates of biogeochemical cycling from in situ measures of dissolved oxygen. Here, we quantified gas ebullition rates in Zostera marina meadows in Virginia, U.S.A. using simple funnel traps and analyzed the oxygen concentration and isotopic composition of the captured gas. Maximum hourly rates of oxygen ebullition (3.0 mmol oxygen m -2 h -1 ) were observed during the coincidence of high irradiance and low tides, particularly in the afternoon when oxygen and temperature maxima occurred. The daily ebullition fluxes (up to 11 mmol oxygen m -2 d -1 ) were roughly equivalent to net primary production rates determined from dissolved oxygen measurements indicating that bubble ebullition can represent a major component of primary production that is not commonly included in ecosystem-scale estimates. Oxygen content comprised 20-40% of the captured bubble gas volume and correlated negatively with its δ 18 O values, consistent with a predominance of mixing between the higher δ 18 O of atmospheric oxygen in equilibrium with seawater and the lower δ 18 O of oxygen derived from photosynthesis. Thus, future studies interested in the metabolism of highly productive, shallow water ecosystems, and particularly those measuring in situ oxygen flux, should not ignore the bubble formation and ebullition processes described here., Competing Interests: None declared., (© 2019 The Authors. Limnology and Oceanography published by Wiley Periodicals, Inc. on behalf of Association for the Sciences of Limnology and Oceanography.)

Reactive oxygen species (ROS) are produced via various photochemical, abiotic, and biological pathways. The low concentration and short lifetime of the ROS superoxide (O 2 •- ) make it challenging to measure in natural systems. Here, we designed, developed, and validated a DIver-operated Submersible Chemiluminescent sensOr (DISCO), the first handheld submersible chemiluminescent sensor. The fluidic system inside DISCO is controlled by two high-precision pumps that introduce sample water and analytical reagents into a mixing cell. The resultant chemiluminescent signal is quantified by a photomultiplier tube, recorded by a miniature onboard computer and monitored in real time via a handheld underwater LED interface. Components are contained within a pressure-bearing housing (max depth 30 m), and an external battery pack supplies power. Laboratory calibrations with filtered seawater verified instrument stability and precision. Field deployment in Cuban coral reefs quantified background seawater-normalized extracellular superoxide concentrations near coral surfaces (0-173 nM) that varied distinctly with coral species. Observations were consistent with previous similar measurements from aquaria and shallow reefs using a standard benchtop system. In situ quantification of superoxide associated with corals was enabled by DISCO, demonstrating the potential application to other shallow water ecosystems and chemical species.

Ammonia-oxidizing archaea (AOA) dominate microbial communities throughout oxic subseafloor sediment deposited over millions of years in the North Atlantic Ocean. Rates of nitrification correlated with the abundance of these dominant AOA populations, whose metabolism is characterized by ammonia oxidation, mixotrophic utilization of organic nitrogen, deamination, and the energetically efficient chemolithoautotrophic hydroxypropionate/hydroxybutyrate carbon fixation cycle. These AOA thus have the potential to couple mixotrophic and chemolithoautotrophic metabolism via mixotrophic deamination of organic nitrogen, followed by oxidation of the regenerated ammonia for additional energy to fuel carbon fixation. This metabolic feature likely reduces energy loss and improves AOA fitness under energy-starved, oxic conditions, thereby allowing them to outcompete other taxa for millions of years.

The benthos in estuarine environments often experiences periods of regularly occurring hypoxic and anoxic conditions, dramatically impacting biogeochemical cycles. How oxygen depletion affects the growth of specific uncultivated microbial populations within these diverse benthic communities, however, remains poorly understood. Here, we applied H 2 18 O quantitative stable isotope probing (qSIP) in order to quantify the growth of diverse, uncultured bacterial populations in response to low oxygen concentrations in estuarine sediments. Over the course of 7- and 28-day incubations with redox conditions spanning from hypoxia to euxinia (sulfidic), 18 O labeling of bacterial populations exhibited different patterns consistent with micro-aerophilic, anaerobic, facultative anaerobic, and aerotolerant anaerobic growth. 18 O-labeled populations displaying anaerobic growth had a significantly non-random phylogenetic distribution, exhibited by numerous clades currently lacking cultured representatives within the Planctomycetes, Actinobacteria, Latescibacteria, Verrucomicrobia, and Acidobacteria. Genes encoding the beta-subunit of the dissimilatory sulfate reductase (dsrB) became 18 O labeled only during euxinic conditions. Sequencing of these 18 O-labeled dsrB genes showed that Acidobacteria were the dominant group of growing sulfate-reducing bacteria, highlighting their importance for sulfur cycling in estuarine sediments. Our findings provide the first experimental constraints on the redox conditions underlying increased growth in several groups of "microbial dark matter", validating hypotheses put forth by earlier metagenomic studies.

Coastal ecosystems host high levels of primary productivity leading to exceptionally dynamic elemental cycling in both water and sediments. In such environments, carbon is rapidly cycled leading to high rates of burial as organic matter and/or high rates of loss to the atmosphere and laterally to the coastal ocean in simpler forms, such as carbon dioxide (CO 2 ) and methane (CH 4 ). To better understand carbon cycling across these heterogeneous environments, new technologies beyond discrete sample collection and analysis are needed to characterize spatial and temporal variability. Here, we describe the ChemYak, an autonomous surface vehicle outfitted with a suite of in situ sensors, developed to achieve large spatial scale chemical mapping of these environments. Dissolved methane and carbon dioxide are measured by a laser spectrometer coupled to a gas extraction unit for continuous quantification during operation. The gas-powered vehicle is capable of rapidly surveying the coastal system with an endurance of up to 10 h at operating speeds in excess of 10 km h -1 . Here, we demonstrate its ability to spatially characterize distributions of CO 2 , CH 4 , oxygen, and nitrate throughout a New England saltmarsh estuary.

The ability of micro-organisms to oxidize manganese (Mn) from Mn(II) to Mn(III/IV) oxides transcends boundaries of biological clade or domain. Many bacteria and fungi oxidize Mn(II) to Mn(III/IV) oxides directly through enzymatic activity or indirectly through the production of reactive oxygen species. Here, we determine the oxygen isotope fractionation factors associated with Mn(II) oxidation via various biotic (bacteria and fungi) and abiotic Mn(II) reaction pathways. As oxygen in Mn(III/IV) oxides may be derived from precursor water and molecular oxygen, we use a twofold approach to determine the isotope fractionation with respect to each oxygen source. Using both 18 O-labeled water and closed-system Rayleigh distillation approaches, we constrain the kinetic isotope fractionation factors associated with O atom incorporation during Mn(II) oxidation to -17.3‰ to -25.9‰ for O 2 and -1.9‰ to +1.8‰ for water. Results demonstrate that stable oxygen isotopes of Mn(III/IV) oxides have potential to distinguish between two main classes of biotic Mn(II) oxidation: direct enzymatic oxidation in which O 2 is the oxidant and indirect enzymatic oxidation in which superoxide is the oxidant. The fraction of Mn(III/IV) oxide-associated oxygen derived from water varies significantly (38%-62%) among these bio-oxides with only weak relationship to Mn oxidation state, suggesting Mn(III) disproportionation may account for differences in the fraction of mineral-bound oxygen from water and O 2 . Additionally, direct incorporation of molecular O 2 suggests that Mn(III/IV) oxides contain a yet untapped proxy of δ18OO2 of environmental O 2 , a parameter reflecting the integrated influence of global respiration, photorespiration, and several other biogeochemical reactions of global significance., (© 2018 John Wiley & Sons Ltd.)

Although increasing atmospheric nitrous oxide (N 2 O) has been linked to nitrogen loading, predicting emissions remains difficult, in part due to challenges in disentangling diverse N 2 O production pathways. As coastal ecosystems are especially impacted by elevated nitrogen, we investigated controls on N 2 O production mechanisms in intertidal sediments using novel isotopic approaches and microsensors in flow-through incubations. Here we show that during incubations with elevated nitrate, increased N 2 O fluxes are not mediated by direct bacterial activity, but instead are largely catalysed by fungal denitrification and/or abiotic reactions (e.g., chemodenitrification). Results of these incubations shed new light on nitrogen cycling complexity and possible factors underlying variability of N 2 O fluxes, driven in part by fungal respiration and/or iron redox cycling. As both processes exhibit N 2 O yields typically far greater than direct bacterial production, these results emphasize their possibly substantial, yet widely overlooked, role in N 2 O fluxes, especially in redox-dynamic sediments of coastal ecosystems.

Manganese (Mn) oxides participate in a range of interactions with organic carbon (OC) that can lead to either carbon degradation or preservation. Here, we examine the abundance and composition of OC associated with biogenic and environmental Mn oxides to elucidate the role of Mn oxides as a reservoir for carbon and their potential for selective partitioning of particular carbon species. Mn oxides precipitated in natural brackish waters and by Mn(II)-oxidizing marine bacteria and terrestrial fungi harbor considerable levels of organic carbon (4.1-17.0 mol OC per kg mineral) compared to ferromanganese cave deposits which contain 1-2 orders of magnitude lower OC. Spectroscopic analyses indicate that the chemical composition of Mn oxide-associated OC from microbial cultures is homogeneous with bacterial Mn oxides hosting primarily proteinaceous carbon and fungal Mn oxides containing both protein- and lipopolysaccharide-like carbon. The bacterial Mn oxide-hosted proteins are involved in both Mn(II) oxidation and metal binding by these bacterial species and could be involved in the mineral nucleation process as well. By comparison, the composition of OC associated with Mn oxides formed in natural settings (brackish waters and particularly in cave ferromanganese rock coatings) is more spatially and chemically heterogeneous. Cave Mn oxide-associated organic material is enriched in aliphatic C, which together with the lower carbon concentrations, points to more extensive microbial or mineral processing of carbon in this system relative to the other systems examined in this study, and as would be expected in oligotrophic cave environments. This study highlights Mn oxides as a reservoir for carbon in varied environments. The presence and in some cases dominance of proteinaceous carbon within the biogenic and natural Mn oxides may contribute to preferential preservation of proteins in sediments and dominance of protein-dependent metabolisms in the subsurface biosphere., (© 2016 John Wiley & Sons Ltd.)

Natural abundance nitrogen and oxygen isotopes of nitrate (δ 15 N NO3 and δ 18 O NO3 ) provide an important tool for evaluating sources and transformations of natural and contaminant nitrate (NO 3 - ) in the environment. Nevertheless, conventional interpretations of NO 3 - isotope distributions appear at odds with patterns emerging from studies of nitrifying and denitrifying bacterial cultures. To resolve this conundrum, we present results from a numerical model of NO 3 - isotope dynamics, demonstrating that deviations in δ 18 O NO3 vs. δ 15 N NO3 from a trajectory of 1 expected for denitrification are explained by isotopic over-printing from coincident NO 3 - production by nitrification and/or anammox. The analysis highlights two driving parameters: (i) the δ 18 O of ambient water and (ii) the relative flux of NO 3 - production under net denitrifying conditions, whether catalyzed aerobically or anaerobically. In agreement with existing analyses, dual isotopic trajectories >1, characteristic of marine denitrifying systems, arise predominantly under elevated rates of NO 2 - reoxidation relative to NO 3 - reduction (>50%) and in association with the elevated δ 18 O of seawater. This result specifically implicates aerobic nitrification as the dominant NO 3 - producing term in marine denitrifying systems, as stoichiometric constraints indicate anammox-based NO 3 - production cannot account for trajectories >1. In contrast, trajectories <1 comprise the majority of model solutions, with those representative of aquifer conditions requiring lower NO 2 - reoxidation fluxes (<15%) and the influence of the lower δ 18 O of freshwater. Accordingly, we suggest that widely observed δ 18 O NO3 vs. δ 15 N NO3 trends in freshwater systems (<1) must result from concurrent NO 3 - production by anammox in anoxic aquifers, a process that has been largely overlooked., Competing Interests: The authors declare no conflict of interest.

Archaeal membrane lipids known as glycerol dibiphytanyl glycerol tetraethers (GDGTs) are the basis of the TEX86 paleotemperature proxy. Because GDGTs preserved in marine sediments are thought to originate mainly from planktonic, ammonia-oxidizing Thaumarchaeota, the basis of the correlation between TEX86 and sea surface temperature (SST) remains unresolved: How does TEX86 predict surface temperatures, when maximum thaumarchaeal activity occurs below the surface mixed layer and TEX86 does not covary with in situ growth temperatures? Here we used isothermal studies of the model thaumarchaeon Nitrosopumilus maritimus SCM1 to investigate how GDGT composition changes in response to ammonia oxidation rate. We used continuous culture methods to avoid potential confounding variables that can be associated with experiments in batch cultures. The results show that the ring index scales inversely (R(2) = 0.82) with ammonia oxidation rate (ϕ), indicating that GDGT cyclization depends on available reducing power. Correspondingly, the TEX86 ratio decreases by an equivalent of 5.4 °C of calculated temperature over a 5.5 fmol·cell(-1)·d(-1) increase in ϕ. This finding reconciles other recent experiments that have identified growth stage and oxygen availability as variables affecting TEX86 Depth profiles from the marine water column show minimum TEX86 values at the depth of maximum nitrification rates, consistent with our chemostat results. Our findings suggest that the TEX86 signal exported from the water column is influenced by the dynamics of ammonia oxidation. Thus, the global TEX86-SST calibration potentially represents a composite of regional correlations based on nutrient dynamics and global correlations based on archaeal community composition and temperature.

A central tenant in microbial biogeochemistry is that microbial metabolisms follow a predictable sequence of terminal electron acceptors based on the energetic yield for the reaction. It is thereby oftentimes assumed that microbial respiration of ferric iron outcompetes sulfate in all but high-sulfate systems, and thus sulfide has little influence on freshwater or terrestrial iron cycling. Observations of sulfate reduction in low-sulfate environments have been attributed to the presumed presence of highly crystalline iron oxides allowing sulfate reduction to be more energetically favored. Here we identified the iron-reducing processes under low-sulfate conditions within columns containing freshwater sediments amended with structurally diverse iron oxides and fermentation products that fuel anaerobic respiration. We show that despite low sulfate concentrations and regardless of iron oxide substrate (ferrihydrite, Al-ferrihydrite, goethite, hematite), sulfidization was a dominant pathway in iron reduction. This process was mediated by (re)cycling of sulfur upon reaction of sulfide and iron oxides to support continued sulfur-based respiration--a cryptic sulfur cycle involving generation and consumption of sulfur intermediates. Although canonical iron respiration was not observed in the sediments amended with the more crystalline iron oxides, iron respiration did become dominant in the presence of ferrihydrite once sulfate was consumed. Thus, despite more favorable energetics, ferrihydrite reduction did not precede sulfate reduction and instead an inverse redox zonation was observed. These findings indicate that sulfur (re)cycling is a dominant force in iron cycling even in low-sulfate systems and in a manner difficult to predict using the classical thermodynamic ladder.

Chemoautotrophic symbionts of deep sea hydrothermal vent tubeworms are known to provide their hosts with all their primary nutrition. While studies have examined how chemoautotrophic symbionts provide the association with nitrogen, fewer have examined if symbiont nitrogen metabolism varies as a function of environmental conditions. Ridgeia piscesae tubeworms flourish at Northeastern Pacific vents, occupy a range of microhabitats, and exhibit a high degree of morphological plasticity [e.g. long-skinny (LS) and short-fat (SF) phenotypes] that may relate to environmental conditions. This plasticity affords an opportunity to examine whether symbiont nitrogen metabolism varies among host phenotypes. LS and SF R. piscesae were recovered from the Axial and Main Endeavour Field hydrothermal vents. Nitrate and ammonium were quantified in Ridgeia blood, and the expression of key nitrogen metabolism genes, as well as stable nitrogen isotope ratios, was quantified in host branchial plume and symbiont-containing tissues. Nitrate and ammonium were abundant in the blood of both phenotypes though environmental ammonium concentrations were, paradoxically, lowest among individuals with the highest blood ammonium. Assimilatory nitrate reductase transcripts were always below detection, though in both LS and SF R. piscesae symbionts, we observed elevated expression of dissimilatory nitrate reductase genes, as well as symbiont and host ammonium assimilation genes. Site-specific differences in expression, along with tissue stable isotope analyses, suggest that LS and SF Ridgeia symbionts are engaged in both dissimilatory nitrate reduction and ammonia assimilation to varying degrees. As such, it appears that environmental conditions -not host phenotype-primarily dictates symbiont nitrogen metabolism., (© 2013 John Wiley & Sons Ltd.)

The vast marine deep biosphere consists of microbial habitats within sediment, pore waters, upper basaltic crust and the fluids that circulate throughout it. A wide range of temperature, pressure, pH, and electron donor and acceptor conditions exists-all of which can combine to affect carbon and nutrient cycling and result in gradients on spatial scales ranging from millimeters to kilometers. Diverse and mostly uncharacterized microorganisms live in these habitats, and potentially play a role in mediating global scale biogeochemical processes. Quantifying the rates at which microbial activity in the subsurface occurs is a challenging endeavor, yet developing an understanding of these rates is essential to determine the impact of subsurface life on Earth's global biogeochemical cycles, and for understanding how microorganisms in these "extreme" environments survive (or even thrive). Here, we synthesize recent advances and discoveries pertaining to microbial activity in the marine deep subsurface, and we highlight topics about which there is still little understanding and suggest potential paths forward to address them. This publication is the result of a workshop held in August 2012 by the NSF-funded Center for Dark Energy Biosphere Investigations (C-DEBI) "theme team" on microbial activity (www.darkenergybiosphere.org).

The capacity to make in situ geo-referenced measurements of methane concentration and stable isotopic composition (δ(13)C(CH4)) would greatly improve our understanding of the distribution and type of methane sources in the environment, allow refined determination of the extent to which microbial production and consumption contributes to methane cycling, and enable the testing of hypotheses about the sensitivity of methane cycling to changes in environmental conditions. In particular, characterizing biogeochemical methane cycling dynamics in the deep ocean is hampered by a number of challenges, especially in environments where high methane concentrations preclude intact recovery of undisturbed samples. To that end, we have developed an in situ analyzer capable of δ(13)C(CH4) measurements in the deep ocean. Here we present data from laboratory and field studies in which we characterize the instrument's analytical capabilities and performance and provide the first in situ stable isotope based characterization of the influence of anaerobic methane oxidation on methane flux from seep sediments. These data illustrate how in situ measurements can permit finer-scale analyses of variations in AOM activity, and facilitate advances in using δ(13)C(CH4) and other isotopic systems to interrogate biogeochemical cycles in the deep sea and other remote or challenging environments.

Iron (Fe) oxides exist in a spectrum of structures in the environment, with ferrihydrite widely considered the most bioavailable phase. Yet, ferrihydrite is unstable and rapidly transforms to more crystalline Fe(III) oxides (e.g., goethite, hematite), which are poorly reduced by model dissimilatory Fe(III)-reducing microorganisms. This begs the question, what processes and microbial groups are responsible for reduction of crystalline Fe(III) oxides within sedimentary environments? Further, how do changes in Fe mineralogy shape oxide-hosted microbial populations? To address these questions, we conducted a large-scale cultivation effort using various Fe(III) oxides (ferrihydrite, goethite, hematite) and carbon substrates (glucose, lactate, acetate) along a dilution gradient to enrich for microbial populations capable of reducing Fe oxides spanning a wide range of crystallinities and reduction potentials. While carbon source was the most important variable shaping community composition within Fe(III)-reducing enrichments, both Fe oxide type and sediment dilution also had a substantial influence. For instance, with acetate as the carbon source, only ferrihydrite enrichments displayed a significant amount of Fe(III) reduction and the well-known dissimilatory metal reducer Geobacter sp. was the dominant organism enriched. In contrast, when glucose and lactate were provided, all three Fe oxides were reduced and reduction coincided with the presence of fermentative (e.g., Enterobacter spp.) and sulfate-reducing bacteria (e.g., Desulfovibrio spp.). Thus, changes in Fe oxide structure and resource availability may shift Fe(III)-reducing communities between dominantly metal-respiring to fermenting and/or sulfate-reducing organisms which are capable of reducing more recalcitrant Fe phases. These findings highlight the need for further targeted investigations into the composition and activity of speciation-directed metal-reducing populations within natural environments.

The anaerobic oxidation of methane (AOM) is a globally significant sink that regulates methane flux from sediments into the oceans and atmosphere. Here we examine mesophilic to thermophilic AOM in hydrothermal sediments recovered from the Middle Valley vent field, on the Juan de Fuca Ridge. Using continuous-flow sediment bioreactors and batch incubations, we characterized (i) the degree to which AOM contributes to net dissolved inorganic carbon flux, (ii) AOM and sulfate reduction (SR) rates as a function of temperature and (iii) the distribution and density of known anaerobic methanotrophs (ANMEs). In sediment bioreactors, inorganic carbon stable isotope mass balances results indicated that AOM accounted for between 16% and 86% of the inorganic carbon produced, underscoring the role of AOM in governing inorganic carbon flux from these sediments. At 90°C, AOM occurred in the absence of SR, demonstrating a striking decoupling of AOM from SR. An abundance of Fe(III)-bearing minerals resembling mixed valent Fe oxides, such as green rust, suggests the potential for a coupling of AOM to Fe(III) reduction in these metalliferous sediments. While SR bacteria were only observed in cooler temperature sediments, ANMEs allied to ANME-1 ribotypes, including a putative ANME-1c group, were found across all temperature regimes and represented a substantial proportion of the archaeal community. In concert, these results extend and reshape our understanding of the nature of high temperature methane biogeochemistry, providing insight into the physiology and ecology of thermophilic anaerobic methanotrophy and suggesting that AOM may play a central role in regulating biological dissolved inorganic carbon fluxes to the deep ocean from the organic-poor, metalliferous sediments of the global mid-ocean ridge hydrothermal vent system., (© 2012 Society for Applied Microbiology and Blackwell Publishing Ltd.)

The discovery of deep-sea hydrothermal vents in 1977 revolutionized our understanding of the energy sources that fuel primary productivity on Earth. Hydrothermal vent ecosystems are dominated by animals that live in symbiosis with chemosynthetic bacteria. So far, only two energy sources have been shown to power chemosynthetic symbioses: reduced sulphur compounds and methane. Using metagenome sequencing, single-gene fluorescence in situ hybridization, immunohistochemistry, shipboard incubations and in situ mass spectrometry, we show here that the symbionts of the hydrothermal vent mussel Bathymodiolus from the Mid-Atlantic Ridge use hydrogen to power primary production. In addition, we show that the symbionts of Bathymodiolus mussels from Pacific vents have hupL, the key gene for hydrogen oxidation. Furthermore, the symbionts of other vent animals such as the tubeworm Riftia pachyptila and the shrimp Rimicaris exoculata also have hupL. We propose that the ability to use hydrogen as an energy source is widespread in hydrothermal vent symbioses, particularly at sites where hydrogen is abundant.

Ammonia oxidation-the microbial oxidation of ammonia to nitrite and the first step in nitrification-plays a central role in nitrogen cycling in coastal and estuarine systems. Nevertheless, questions remain regarding the connection between this biogeochemical process and the diversity and abundance of the mediating microbial community. In this study, we measured nutrient fluxes and rates of sediment nitrification in conjunction with the diversity and abundance of ammonia-oxidizing archaea (AOA) and ammonia-oxidizing betaproteobacteria (β-AOB). Sediments were examined from four sites in Elkhorn Slough, a small agriculturally impacted coastal California estuary that opens into Monterey Bay. Using an intact sediment core flowthrough incubation system, we observed significant correlations among NO(3)(-), NO(2)(-), NH(4)(+), and PO(4)(3+) fluxes, indicating a tight coupling of sediment biogeochemical processes. (15)N-based measurements of nitrification rates revealed higher rates at the less impacted, lower-nutrient sites than at the more heavily impacted, nutrient-rich sites. Quantitative PCR analyses revealed that β-AOB amoA (encoding ammonia monooxygenase subunit A) gene copies outnumbered AOA amoA gene copies by factors ranging from 2- to 236-fold across the four sites. Sites with high nitrification rates primarily contained marine/estuarine Nitrosospira-like bacterial amoA sequences and phylogenetically diverse archaeal amoA sequences. Sites with low nitrification rates were dominated by estuarine Nitrosomonas-like amoA sequences and archaeal amoA sequences similar to those previously described in soils. This is the first report measuring AOA and β-AOB amoA abundance in conjunction with (15)N-based nitrification rates in estuary sediments.

Global inputs of NO(x) are dominated by fossil fuel combustion from both stationary and vehicular sources and far exceed natural NO(x) sources. However, elucidating NO(x) sources to any given location remains a difficult challenge, despite the need for this information to develop sound regulatory and mitigation strategies. We present results from a regional-scale study of nitrogen isotopes (delta15N) in wet nitrate deposition across 33 sites in the midwestern and northeastern U.S. We demonstrate that spatial variations in delta15N are strongly correlated with NO(x) emissions from surrounding stationary sources and additionally that delta15N is more strongly correlated with surrounding stationary source NO(x) emissions than pH, SO4(2-), or NO3- concentrations. Although emission inventories indicate that vehicle emissions are the dominant NO(x) source in the eastern U.S., our results suggest that wet NO3- deposition at sites in this study is strongly associated with NO(x) emissions from stationary sources. This suggests that large areas of the landscape potentially receive atmospheric NO(y) deposition inputs in excess of what one would infer from existing monitoring data alone. Moreover, we determined that spatial patterns in delta15N values are a robust indicator of stationary NO(x) contributions to wet NO3- deposition and hence a valuable complement to existing tools for assessing relationships between NO3- deposition, regional emission inventories, and for evaluating progress toward NO(x) reduction goals.

Sewage effluent, storm runoff, discharge from polluted rivers, and inputs of groundwater have all been suggested as potential sources of land derived nutrients into Hanalei Bay, Kauai. We determined the nitrogen isotopic signatures (delta(15)N) of different nitrate sources to Hanalei Bay along with the isotopic signature recorded by 11 species of macroalgal collected in the Bay. The macroalgae integrate the isotopic signatures of the nitrate sources over time, thus these data along with the nitrate to dissolved inorganic phosphate molar ratios (N:P) of the macroalgae were used to determine the major nitrate source to the bay ecosystem and which of the macro-nutrients is limiting algae growth, respectively. Relatively low delta(15)N values (average -0.5% per hundred) were observed in all algae collected throughout the Bay; implicating fertilizer, rather than domestic sewage, as an important external source of nitrogen to the coastal water around Hanalei. The N:P ratio in the algae compared to the ratio in the Bay waters imply that the Hanalei Bay coastal ecosystem is nitrogen limited and thus, increased nitrogen input may potentially impact this coastal ecosystem and specifically the coral reefs in the Bay. Identifying the major source of nutrient loading to the Bay is important for risk assessment and potential remediation plans.

As part of a multi-agency study of alligator health, 28 American alligators (Alligator mississippiensis) were captured along a transect through the Florida Everglades in 1999. Liver and tail muscle tissues were sampled and analyzed on a wet weight basis for total mercury (THg) using cold-vapor atomic absorption spectrophotometry. All tissues had detectable concentrations of THg that ranged from 0.6 to 17 mg/kg in liver and from 0.1 to 1.8 mg/kg in tail muscle. THg was more concentrated in liver tissue than tail muscle, but levels were highly correlated between tissues. THg concentrations in tissue differed significantly among locations, with animals from Everglades National Park (ENP) having mean concentrations of THg in liver (10.4 mg/kg) and tail muscle (1.2 mg/kg) that were two-fold higher than basin-wide averages (4.9 and 0.64 mg/kg, respectively). The reasons for higher contamination of ENP alligators were unclear and could not be explained by differences in sex, length, weight or animal age. While delta15N values were positively correlated with THg concentrations in tail muscle, spatial patterns in isotopic composition did not explain the elevated THg levels in ENP alligators. Therefore, it appears that ENP alligators were more highly exposed to mercury in their environment than individuals in other areas. Comparisons to a previous survey by Yanochko et al. [Arch Environ Contam Toxicol 32 (1997) 323] suggest that mercury levels have declined in some Everglades alligators since 1994.