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The nitrogen (N) and oxygen (O) stable isotope analysis of dissolved nitrous oxide (N2O) can provide important constraints on the sources and cycling of N2O in aquatic environments. The isotopic composition of aqueous N2O, both in field (natural abundance) or experimental (15N‐labeling) samples, however, may be altered by abiotic reactions involving nitrite (NO2−) or hydroxylamine (NH2OH) and microbial activity during sample storage, if samples are not adequately preserved. Here we tested five different preservatives, mercuric chloride (HgCl2), copper sulfate (CuSO4), zinc chloride (ZnCl2), hydrochloric acid (HCl) mixed with sulfamic acid (SFA), and sodium hydroxide (NaOH), for fixing natural water samples from an estuary and a lake with different NO2− concentrations over a range of different storage times for N2O analyses. ZnCl2 and CuSO4 decreased the pH, and led to abiotic N2O production from 15NO2−, shifting the N2O isotopic composition significantly. Removal of NO2− with a mixture of SFA and HCl did not always prevent the alteration of the original N and O isotope composition of N2O, confirming the requirement for complete NO2− removal, and underscoring the biasing effects of NO2− at very low pH, even at trace levels. At low NO2− background, HgCl2 preservation led to robust and accurate results. However, in light of its toxicity and bioaccumulation potential in food webs, this fixative should be avoided. The addition of NaOH reduced abiotic side reactions involving NO2−, and yielded the most reliable and reproducible results for N2O concentration and isotope ratio measurements across tested settings and storage times. [ABSTRACT FROM AUTHOR]

Nitrogen (N) bioavailability affects phytoplankton growth and primary production in the aquatic environment. N bioavailability is partly determined by biological N cycling processes that either transform N species or remove fixed N. Reliable estimates of their kinetic parameters can help understand the distribution of N cycling processes. However, available estimates of kinetic parameters are often derived from microbial isolates and may not be representative of the natural environment. Observations are particularly lacking in estuarine and coastal waters. We conducted isotope tracer addition incubations to evaluate substrate affinities of nitrification, denitrification and anammox in the Chesapeake Bay water column. The half‐saturation constant for ammonia oxidation ranged from 0.38 to 0.75 μM ammonium, substantially higher than observed in the open oceans. Half‐saturation constants for denitrification—0.92–1.86 μM nitrite or 1.15 μM nitrate—were within the lower end or less than those reported for other aquatic environments and for denitrifier isolates. Interestingly, water column denitrification potential was comparable to that of sedimentary denitrification, highlighting the contribution of the water column to N removal during anoxia. Mostly undetectable anammox rates prevented us from deriving the half‐saturation constants, suggesting a low affinity of anammox. Using these substrate kinetics, we were able to predict in situ N cycling rates and explain the vertical distribution of N nutrient concentrations. Our newly derived substrate kinetics parameters can be useful for improving model representation of N nutrient dynamics in estuarine and coastal waters, which is critical for assessing the ecosystem productivity and function. [ABSTRACT FROM AUTHOR]

The underlying mechanism for sustaining the high productivity of coral reefs in nutrient deplete water, known as the Darwin Paradox, has long been debated. The input of new nitrogen, for example, derived from N2 fixation, and efficient nitrogen recycling are hypothesized as two critical factors in resolving the paradox. However, the source of new nitrogen and its connection to nitrogen recycling in the coral reef system remain poorly understood. We measured N2 fixation rates and investigated the diazotroph community in different components of a tropical coral reef system. Our results show that the reef‐building crustose coralline algae demonstrate a significantly higher N2 fixation rate than the overlying water or the coral holobiont, representing a large source of N2 fixation to the system. The associated diazotroph community was dominated by the non‐cyanobacterial Rhizobiales. 15N2‐based pulse‐chase incubation experiments revealed ~ 50% of the diazotroph‐derived nitrogen was released from the crustose coralline algae holobiont to the overlying water and coral holobiont within 48 h, which was five fold higher than the fraction of nitrogen released from nitrate‐based assimilation by crustose coralline algae. Furthermore, the released diazotroph‐derived nitrogen was rapidly transferred to the coral holobiont through grazing and was subsequently released back to the overlying water as dissolved organic nitrogen, revealing an efficient pathway that connects the new nitrogen input with the nitrogen recycling in the coral reef ecosystem. Our results provide new insights into the sources and fates of new nitrogen in the coral reefs, helping to resolve the Darwin Paradox in this unique ecosystem. [ABSTRACT FROM AUTHOR]

Fixed nitrogen limits primary productivity in most areas of the surface ocean. Nitrite oxidation is the main source of nitrate, the most abundant form of inorganic fixed nitrogen. Even though known as an aerobic process, nitrite oxidation is not always stimulated by increased oxygen concentration, and nitrite oxidation occurs in layers of oxygen minimum zones (OMZs) where oxygen is not detectable. Nitrite‐oxidizing bacteria, known since their original isolation as aerobes, were also detected in these layers. Whether and how nitrite oxidation is occurring in the anoxic seawater is debated. Here, we reassess recent advances in marine nitrite oxidation in OMZ regions using previous work and new data sets we collected in two Pacific OMZs. We analyze the complex relationship between nitrite oxidation and oxygen. We discuss potential mechanisms explaining nitrite oxidation in different layers of OMZs based on recent findings and propose future directions to resolve the controversial question of apparently anaerobic nitrite oxidation in anoxic layers. Plain Language Summary: No organism can survive without nitrogen. The scarcity of fixed nitrogen in large surface areas of the ocean limits the capacity of phytoplankton to fix carbon dioxide. Oxygen minimum zones (OMZs) are critical oceanic regions for marine nitrogen inventory because the full spectrum of oxygen conditions in OMZs allows the retention and the loss of fixed nitrogen. The anoxic layer in OMZs was thought to host only anaerobic nitrogen loss processes. However, nitrite oxidation, conventionally known as an aerobic process that retains fixed nitrogen, was also detected in the anoxic zone. We revisit the complex relationship between nitrite oxidation and oxygen, and present mechanisms explaining nitrite oxidation in different layers of the ocean. We also chart a way forward for deciphering the mystery of anaerobic nitrite oxidation. Key Points: The detection of nitrite oxidation in anoxic seawater suggests the necessity to reevaluate our understanding of the nitrogen budgetDifferent mechanisms explaining nitrite oxidation in different oceanic layers are discussedThe complex relationship between nitrite oxidation and O2 indicates the importance of rigorous O2 control during experimental manipulations [ABSTRACT FROM AUTHOR]

Marine oxygen‐deficient zones represent a natural source of nitrous oxide (N2O), a potent greenhouse gas and ozone‐depleting agent. To investigate controls on N2O production, the responses of ammonia oxidation (AO) to nitrite (NO2−) and N2O with respect to oxygen (O2), ammonium (NH4+) and NO2− concentrations were evaluated using N−NH4+15 tracer incubations in the Eastern Tropical North Pacific. Within the oxycline, additions of NH4+ and O2 stimulated N2O production according to Michaelis–Menten kinetics, indicating that both substrates were limiting, and that N2O production, even if the exact mechanisms remain uncertain, is mediated by predictable kinetics. Low half‐saturation constants for NH4+ (12–28 nM) and O2 (460 ± 130 nM) during N2O production indicate that AO communities are well adapted to low concentrations of both substrates. Hybrid N2O formation (i.e., from one NH4+15 and one unlabeled nitrogen (N) source, e.g., NO2−, NO) accounted for ~ 90% of the N2O production from NH4+ and was robust across the different O2, NO2−, and NH4+ conditions. Lack of response to variable substrate concentrations implies that the unlabeled N source was not limiting for N2O production. Although both O2 and NH4+ were key modulators of N2O production rates, N2O yield (N2O produced per NO2− produced) seemed to be controlled solely by O2. The N2O yield increased when O2 concentrations dropped below the half‐saturation concentration for AO to NO2− (< 1.4 μM), the range where NO2− production decreased faster than N2O production. Our study shows that O2 control on N2O yield from AO is robust across stations and depths. [ABSTRACT FROM AUTHOR]

The coastal ocean and marginal sea play a disproportionally important role in the release of nitrous oxide (N2O) into the atmosphere. The spatial and temporal distribution of N2O in these important source regions remains highly uncertain due to the scarcity of N2O measurements. Here we present a large data set of N2O concentrations and fluxes obtained from 10 cruises covering four seasons in the Northern South China Sea (NSCS). The study area is overall a net source of atmospheric N2O with an annual flux of 1.9 ± 1.2 × 108, 0.8 ± 0.5 × 108 and 1.2 ± 0.7 × 108 mol N2O yr−1 in the shelf, slope and basin regions, respectively. In terms of global warming potentials, the N2O emissions offset 27.8% of the CO2 sink on the shelf, and are equivalent to 3.5 and 0.2‐fold of the CO2 emission in the slope and basin of the NSCS. On the seasonal time scale, N2O flux was significantly higher in autumn and winter than in the warm seasons. The spatial variability was contrastingly less pronounced. The seasonality of N2O distribution in the shelf region was modulated by the riverine discharge, while intrusion of the Kuroshio Current exerted profound control on N2O distribution in the open waters of the NSCS. The variable relationships between N2O excess, apparent oxygen utilization, and nitrate in the shelf, NSCS basin and the Luzon Strait indicated a regional difference in N2O cycling pathways along with the impact of water mass mixing. Our study establishes a robust baseline to understand N2O distribution and flux in the NSCS. Key Points: N2O dynamics demonstrate significant seasonal, but much less pronounced spatial variations in the Northern South China SeaAnnual N2O emmision offset 28% of the CO2 sink on the shelf, and are equivalent to 249% and 23% of the CO2 emission in the slope and basinRiverine discharge and Kuroshio Current intrusions are the main drivers of N2O dynamics in the Northern South China Sea [ABSTRACT FROM AUTHOR]

Estuaries at the global scale are significant but highly uncertain sources of atmospheric nitrous oxide (N2O), which is an intense greenhouse gas and ozone depletion agent. As the largest estuary in the United States, the Chesapeake Bay is suggested to be a spatially and temporally variable source and sink of N2O. However, limited observations of N2O cycling preclude us from estimating and predicting its net N2O flux. To improve our mechanistic understanding of the processes that control the N2O flux at the point of production, we applied multiple 15N tracers (NH4+15, 15N‐urea, NO2−15, and NO3−15) to separately track N2O production from nitrification and denitrification under in situ and manipulated O2 concentrations in the Chesapeake Bay. Nitrification was the major N2O production pathway in oxic waters (up to 7.5 nmol N2O L−1 d−1). In contrast, denitrification dominated N2O production from hypoxic/anoxic waters (up to 20 nmol N2O L−1 d−1). N2O production from urea was observed for the first time in estuarine waters. The contribution from urea was small, but interestingly, showed a depth pattern distinct from other N2O precursors. Experimentally lowering the O2 concentration substantially enhanced N2O production. Therefore, the expansion of hypoxic and anoxic zones in the Chesapeake Bay under climate change as suggested by some climate models may favor the production of N2O, potentially providing positive feedback on warming. Overall, our study provides mechanistic constraints on N2O dynamics that could benefit modeling studies to better estimate the N2O flux in the Chesapeake Bay and other coastal environments. [ABSTRACT FROM AUTHOR]

Central to the Southern Ocean's role in setting atmospheric CO2 is the seasonal alternation between upward mixing of nutrients and their subsequent consumption by phytoplankton. Active nutrient cycling within the mixed layer, including the release of ammonium (NH4+) and its removal by phytoplankton and nitrifiers, also affects Southern Ocean CO2 drawdown, yet remains poorly understood. We conducted kinetics experiments across the Southern Ocean south of Africa to investigate the dependence of NH4+ uptake (summer, winter) and oxidation (winter) on NH4+ concentration. NH4+ uptake followed a Michaelis–Menten function in both seasons, with the maximum rate (Vmax) decreasing poleward, apparently controlled mainly by light in winter and temperature in summer. The half‐saturation constant (Km) increased poleward with increasing ambient NH4+ ([NH4+]amb) and was threefold higher in winter (150–405 nM) than in summer (41–115 nM), suggesting that summertime phytoplankton are adapted to low‐NH4+ conditions while winter communities typically receive a higher NH4+ supply. NH4+ oxidation showed a high affinity for NH4+ (Km = 28–137 nM), suggesting a dominant role for ammonia‐oxidizing archaea, and followed a Michaelis–Menten curve only when [NH4+]amb was ≤ 90 nM. Vmax was near‐constant across the region regardless of [NH4+]amb, temperature, or light. From coincident mixed‐layer NH4+ oxidation and iron measurements, we hypothesize that iron availability may (co‐)limit the Vmax of NH4+ oxidation. If verified, this suggestion has implications for models that parameterize nitrification as a linear function of [NH4+]amb. Additionally, iron depletion may limit the role of mixed‐layer nitrification, which is dominant in the winter Southern Ocean, in offsetting phytoplankton CO2 drawdown annually. [ABSTRACT FROM AUTHOR]

As a key intermediate in the nitrogen cycle, nitrite is involved in multiple biological pathways that regulate the distribution and availability of nitrogen in the ocean. In the oligotrophic gyres, nitrite accumulates near the base of the euphotic zone, demonstrating a subsurface maximum, termed the primary nitrite maximum; while in subpolar regions, nitrite concentrations are elevated in the near-surface ocean. As yet, the mechanisms controlling this meridional pattern remain unclear. Here, we present vertically resolved profiles of rates of nitrite production and consumption extending from the Subtropical Gyre to the Subarctic Front in the North Pacific Ocean. Our results indicate that the latitudinal distributions of nitrite across this basin are influenced by variations in phytoplankton-nitrifier interactions. In the well-lit oligotrophic surface, phytoplankton dominates rapid nitrite cycling via coupled release and re-assimilation; below the euphotic zone, diminished light stress on nitrite oxidizers results in rapid turnover and limits nitrite. By contrast, in subpolar regions where nitrate concentrations are elevated in the euphotic zone, nitrite is released during assimilative nitrate reduction and competition between phytoplankton and nitrifiers for ammonium is relaxed, facilitating ammonia oxidation. These processes, together with differential light sensitivities of ammonia and nitrite oxidizers, allow net accumulation of nitrite. Furthermore, we demonstrate a substantial contribution of urea oxidation in forming the primary nitrite maximum and balancing the two steps of marine nitrification. Our findings reveal physical- biological interactive controls on nitrite cycling and distributions in the ocean and help disentangle the complex effect of phytoplankton-microbe interactions on marine nitrogen biogeochemistry. [ABSTRACT FROM AUTHOR]

Net primary production (NPP) fueled by nitrate is often equated with carbon export, providing a metric for CO2 removal to the deep ocean. This "new production paradigm" assumes that nitrification, the oxidation of regenerated ammonium to nitrate, is negligible in the sunlit upper ocean. While surface layer nitrification has been measured in other oceanic regions, very few data exist for the Southern Ocean. We measured NPP, nitrogen (N) uptake, and nitrification in the upper 200 m across the Atlantic Southern Ocean in winter and summer. Rates of winter mixed‐layer nitrate uptake were low, while ammonium uptake was surprisingly high. NPP was also low, such that NPP and total N (nitrate+ammonium) uptake were decoupled; we attribute this to ammonium consumption by heterotrophic bacteria. By contrast, NPP and total N uptake were strongly coupled in summer except at two stations where an additional regenerated N source, likely dissolved organic N, apparently supported 30–45% of NPP. Summertime nitrate uptake rates were fairly high and nitrate fueled >50% of NPP, indicating the potential for significant carbon export. Nitrification supplied <10% of the nitrate consumed in summertime surface waters, while in winter, mixed‐layer nitrification was on average 16 times higher than nitrate uptake. Despite the near‐zero nitrification rates measured in the summer mixed layer, the classically defined f ratio does not well‐represent Southern Ocean carbon export potential annually. This is because some fraction of the nitrate regenerated in the winter mixed layer is likely supplied to phytoplankton in summer; its consumption cannot, therefore, be equated with export. Key Points: The dominant biological process acting on the surface Southern Ocean nitrate pool is assimilation in summer and nitrification in winterPrimary production and nitrogen uptake are coupled in summer but not winter, likely due to ammonium assimilation by heterotrophic bacteria15N‐based estimates of the f ratio do not well‐represent Southern Ocean carbon export potential on an annual basis [ABSTRACT FROM AUTHOR]

Prochlorococcus and Synechococcus are the most abundant free‐living photosynthetic microorganisms in the ocean. Uncultivated lineages of these picocyanobacteria also thrive in the dimly illuminated upper part of oxygen‐deficient zones (ODZs), where an important portion of ocean nitrogen (N) loss takes place via denitrification and anaerobic ammonium oxidation. Recent metagenomic studies revealed that ODZ Prochlorococcus have the genetic potential for using different N forms, including nitrate and nitrite, uncommon N sources for Prochlorococcus, but common for Synechococcus. To determine which N sources ODZ picocyanobacteria are actually using in nature, the cellular 15N natural abundance (δ15N) and assimilation rates of different N compounds were determined using cell sorting by flow cytometry and mass spectrometry. The natural δ15N of the ODZ Prochlorococcus varied from −4.0‰ to 13.0‰ (n = 9), with 50% of the values in the range of −2.1–2.6‰. While the highest values suggest nitrate use, most observations indicate the use of nitrite, ammonium, or a mixture of N sources. Meanwhile, incubation experiments revealed potential assimilation rates of ammonium and urea in the same order of magnitude as that expected for total N in several environments including ODZs, whereas rates of nitrite and nitrate assimilation were very low. Our results thus indicate that reduced forms of N and nitrite are the dominant sources for ODZ picocyanobacteria, although nitrate might be important on some occasions. ODZ picocyanobacteria might thus represent potential competitors with anammox bacteria for ammonium and nitrite, with ammonia‐oxidizing archaea for ammonium, and with nitrite‐oxidizing bacteria for nitrite. [ABSTRACT FROM AUTHOR]

Recent work has suggested that the oxygen deficient zone (ODZ) and overlying surface waters of the eastern tropical South Pacific (ETSP) is a potential niche for dinitrogen (N2) fixation. Rates of dinitrogen fixation were measured in the ETSP above and within the ODZ in July 2013 using a modified 15N2 bubble addition method, wherein a bubble was added, mixed, and then removed, and the isotopic enrichment of the dissolved N2 was measured directly for each incubation. N2 fixation rates in the euphotic zone ranged from below detection to 3.9 nmol L−1 d−1 and were below detection at all depths surveyed within the ODZ. Depth‐integrated rates ranged from below detection to 289.7 μmol m−2 d−1. DNA and RNA of diverse nifH genes were detected at both surface waters and in the ODZ. However, the results of this study suggest that N2 fixation rates were low and contribute little to N cycling in the ETSP. [ABSTRACT FROM AUTHOR]

The ocean is estimated to contribute up to ~20% of global fluxes of atmospheric nitrous oxide (N2O), an important greenhouse gas and ozone depletion agent. Marine oxygen minimum zones contribute disproportionately to this flux. To further understand the partition of nitrification and denitrification and their environmental controls on marine N2O fluxes, we report new relationships between oxygen concentration and rates of N2O production from nitrification and denitrification directly measured with 15N tracers in the Eastern Tropical Pacific. Highest N2O production rates occurred near the oxic‐anoxic interface, where there is strong potential for N2O efflux to the atmosphere. The dominant N2O source in oxygen minimum zones was nitrate reduction, the rates of which were 1 to 2 orders of magnitude higher than those of ammonium oxidation. The presence of oxygen significantly inhibited the production of N2O from both nitrification and denitrification. These experimental data provide new constraints to a multicomponent global ocean biogeochemical model, which yielded annual oceanic N2O efflux of 1.7–4.4 Tg‐N (median 2.8 Tg‐N, 1 Tg = 1012 g), with denitrification contributing 20% to the oceanic flux. Thus, denitrification should be viewed as a net N2O production pathway in the marine environment. Key Points: New quantitative relationships between oxygen and nitrous oxide production are derived from direct measurements in low‐oxygen oceansThe presence of oxygen reduces nitrous oxide production via nitrification and denitrificationThe latest biogeochemical model incorporating the field measurements and relationships provides new estimate of global marine nitrous oxide flux [ABSTRACT FROM AUTHOR]

Abstract: Nitrification, the oxidation of ammonium to nitrite and then nitrate, is often discounted as a source of nitrate in euphotic zone waters due to photoinhibition of nitrifying microorganisms and/or competition for ammonium with phytoplankton. However, there have also been counterarguments that nitrification represents a significant “regenerated” nitrate source to phytoplankton, augmenting the “new” nitrate supplied via physical processes. If nitrification is an appreciable nitrate source in the euphotic zone, then the assumption of a balance between nitrate uptake and organic matter export will overestimate export production. We investigated the relative importance of nitrification and nitrate uptake in the subarctic North Atlantic in late spring and late summer. The rates and vertical distributions of primary production, nitrogen uptake, and ammonium and nitrite oxidation were determined through isotope tracer experiments and the distributions of the nitrogen and oxygen isotopes of nitrate. In surface waters, ammonium and nitrite oxidation rates were low, representing an average of 5.2% and 2.5% of total euphotic zone nitrate uptake, respectively. The nitrogen and oxygen isotopes of nitrate confirmed that nitrification was not significant within the euphotic zone. Comparison of the rates of nitrogen uptake and primary production showed that while springtime phytoplankton growth could be fully supported by new nitrate and recycled ammonium, up to 50% of summertime productivity was likely fueled by dissolved organic nitrogen (DON). Uptake of DON implies that the fraction of primary production exported from surface waters in the late summer was significantly lower than the measured nitrate and ammonium uptake rates suggest. [ABSTRACT FROM AUTHOR]

Molecular analysis of dissimilatory nitrite reductase genes ( nirS) was conducted using a customized microarray containing 165 nirS probes (archetypes) to identify members of sedimentary denitrifying communities. The goal of this study was to examine denitrifying community responses to changing environmental variables over spatial and temporal scales in the New River Estuary (NRE), NC, USA. Multivariate statistical analyses revealed three denitrifier assemblages and uncovered 'generalist' and 'specialist' archetypes based on the distribution of archetypes within these assemblages. Generalists, archetypes detected in all samples during at least one season, were commonly world-wide found in estuarine and marine ecosystems, comprised 8%-29% of the abundant NRE archetypes. Archetypes found in a particular site, 'specialists', were found to co-vary based on site specific conditions. Archetypes specific to the lower estuary in winter were designated Cluster I and significantly correlated by sediment Chl a and porewater Fe2+. A combination of specialist and more widely distributed archetypes formed Clusters II and III, which separated based on salinity and porewater H2S respectively. The co-occurrence of archetypes correlated with different environmental conditions highlights the importance of habitat type and niche differentiation among nirS-type denitrifying communities and supports the essential role of individual community members in overall ecosystem function. [ABSTRACT FROM AUTHOR]

To investigate the controls on N2 fixation and the role of the Atlantic in the global ocean's fixed nitrogen (N) budget, Atlantic N2 fixation is calculated by combining meridional nitrate fluxes across World Ocean Circulation Experiment sections with observed nitrate 15N/14N differences between northward and southward transported nitrate. N2 fixation inputs of 27.1 ± 4.3 Tg N/yr and 3.0 ± 0.5 Tg N/yr are estimated north of 11°S and 24°N, respectively. That is, ~90% of the N2 fixation in the Atlantic north of 11°S occurs south of 24°N in a region with upwelling that imports phosphorus (P) in excess of N relative to phytoplankton requirements. This suggests that, under the modern iron-rich conditions of the equatorial and North Atlantic, N2 fixation occurs predominantly in response to P-bearing, N-poor conditions. We estimate a N2 fixation rate of 30.5 ± 4.9 Tg N/yr north of 30°S, implying only 3 Tg N/yr between 30° and 11°S, despite evidence of P-bearing, N-poor surface waters in this region as well; this is consistent with iron limitation of N2 fixation in the South Atlantic. Since the ocean flows through the Atlantic surface in <2,500 years, similar to the residence time of oceanic fixed N, Atlantic N2 fixation can stabilize the N-to-P ratio of the global ocean. However, the calculated rate of Atlantic N2 fixation is a small fraction of global ocean estimates for either N2 fixation or fixed N loss. This suggests that, in the modern ocean, an approximate balance between N loss and N2 fixation is achieved within the combined Indian and Pacific basins. [ABSTRACT FROM AUTHOR]

Nitrite oxidation is an essential step in transformations of fixed nitrogen. The physiology of nitrite oxidizing bacteria (NOB) implies that the rates of nitrite oxidation should be controlled by concentration of their substrate, nitrite, and the terminal electron acceptor, oxygen. The sensitivities of nitrite oxidation to oxygen and nitrite concentrations were investigated using 15N tracer incubations in the Eastern Tropical North Pacific. Nitrite stimulated nitrite oxidation under low in situ nitrite conditions, following Michaelis-Menten kinetics, indicating that nitrite was the limiting substrate. The nitrite half-saturation constant ( K s = 0.254 ± 0.161 μM) was 1-3 orders of magnitude lower than in cultivated NOB, indicating higher affinity of marine NOB for nitrite. The highest rates of nitrite oxidation were measured in the oxygen depleted zone (ODZ), and were partially inhibited by additions of oxygen. This oxygen sensitivity suggests that ODZ specialist NOB, adapted to low-oxygen conditions, are responsible for apparently anaerobic nitrite oxidation. [ABSTRACT FROM AUTHOR]

The Eastern Tropical South Pacific is one of the three major oxygen deficient zones (ODZs) in the global ocean and is responsible for approximately one third of marine water column nitrogen loss. It is the best studied of the ODZs and, like the others, features a broad nitrite maximum across the low oxygen layer. How the microbial processes that produce and consume nitrite in anoxic waters interact to sustain this feature is unknown. Here we used 15N-tracer experiments to disentangle five of the biologically mediated processes that control the nitrite pool, including a high-resolution profile of nitrogen loss rates. Nitrate reduction to nitrite likely depended on organic matter fluxes, but the organic matter did not drive detectable rates of denitrification to N2. However, multiple lines of evidence show that denitrification is important in shaping the biogeochemistry of this ODZ. Significant rates of anaerobic nitrite oxidation at the ODZ boundaries were also measured. Iodate was a potential oxidant that could support part of this nitrite consumption pathway. We additionally observed N2 production from labeled cyanate and postulate that anammox bacteria have the ability to harness cyanate as another form of reduced nitrogen rather than relying solely on ammonification of complex organic matter. The balance of the five anaerobic rates measured-anammox, denitrification, nitrate reduction, nitrite oxidation, and dissimilatory nitrite reduction to ammonium-is sufficient to reproduce broadly the observed nitrite and nitrate profiles in a simple one-dimensional model but requires an additional source of reduced nitrogen to the deeper ODZ to avoid ammonium overconsumption. [ABSTRACT FROM AUTHOR]

Marine oxygen deficient zones (ODZs) have long been identified as sites of fixed nitrogen (N) loss. However, the mechanisms and rates of N loss have been debated, and traditional methods for measuring these rates are labor-intensive and may miss hot spots in spatially and temporally variable environments. Here we estimate rates of heterotrophic nitrate reduction, heterotrophic nitrite reduction (denitrification), nitrite oxidation, and anaerobic ammonium oxidation (anammox) at a coastal site in the eastern tropical South Pacific (ETSP) ODZ based on high-resolution concentration and natural abundance stable isotope measurements of nitrate (NO3−) and nitrite (NO2−). These measurements were used to estimate process rates using a two-step inverse modeling approach. The modeled rates were sensitive to assumed isotope effects for NO3− reduction and NO2− oxidation. Nevertheless, we addressed two questions surrounding the fates of NO2− in the ODZ: (1) Is NO2− being primarily reduced to N2 or oxidized to NO3− in the ODZ? and (2) what are the contributions of anammox and denitrification to NO2− removal? Depth-integrated rates from the model suggest that 72-88% of the NO2− produced in the ODZ was oxidized back to NO3−, while 12-28% of NO2− was reduced to N2. Furthermore, our model suggested that 36-74% of NO2− loss was due to anammox, with the remainder due to denitrification. These model results generally agreed with previously measured rates, though with a large range of uncertainty, and they provide a long-term integrated view that compliments incubation experiments to obtain a broader picture of N cycling in ODZs. [ABSTRACT FROM AUTHOR]

Nitrification plays a key role in the marine nitrogen (N) cycle, including in oceanic oxygen minimum zones (OMZs), which are hot spots for denitrification and anaerobic ammonia oxidation (anammox). Recent evidence suggests that nitrification links the source (remineralized organic matter) and sink (denitrification and anammox) of fixed N directly in the steep oxycline in the OMZs. We performed shipboard incubations with 15N tracers to characterize the depth distribution of nitrification in the Eastern Tropical North Pacific (ETNP). Additional experiments were conducted to investigate photoinhibition. Allylthiourea (ATU) was used to distinguish the contribution of archaeal and bacterial ammonia oxidation. The abundance of archaeal and β-proteobacterial ammonia monooxygenase gene subunit A ( amoA) was determined by quantitative polymerase chain reaction. The rates of ammonia and nitrite oxidation showed distinct subsurface maxima, with the latter slightly deeper than the former. The ammonia oxidation maximum coincided with the primary nitrite concentration maximum, archaeal amoA gene maximum, and the subsurface nitrous oxide maximum. Negligible rates of ammonia oxidation were found at anoxic depths, where high rates of nitrite oxidation were measured. Archaeal amoA gene abundance was generally 1 to 2 orders of magnitude higher than bacterial amoA gene abundance, and inhibition of ammonia-oxidizing bacteria with 10 μM ATU did not affect ammonia oxidation rates, indicating the dominance of archaea in ammonia oxidation. These results depict highly dynamic activities of ammonia and nitrite oxidation in the oxycline of the ETNP OMZ. [ABSTRACT FROM AUTHOR]

The Gulf of Mexico is affected by hurricanes and suffers seasonal hypoxia. The Deepwater Horizon oil spill impacted every trophic level in the coastal region. Despite their importance in bioremediation and biogeochemical cycles, it is difficult to predict the responses of microbial communities to physical and anthropogenic disturbances. Here, we quantify sediment ammonia-oxidizing archaeal ( AOA) community diversity, resistance and resilience, and important geochemical factors after major hurricanes and the oil spill. Dominant AOA archetypes correlated with different geochemical factors, suggesting that different AOA are constrained by distinct parameters. Diversity was lowest after the hurricanes, showing weak resistance to physical disturbances. However, diversity was highest during the oil spill and coincided with a community shift, suggesting a new alternative stable state sustained for at least 1 year. The new AOA community was not significantly different from that at the spill site 1 year after the spill. This sustained shift in nitrifier community structure may be a result of oil exposure. [ABSTRACT FROM AUTHOR]

The Sargasso Sea is characterized by strong summertime stratification that is thought to drive oligotrophy, but export production is surprisingly similar to that of high-latitude regions with ample major nutrient supply. Here we use the summer-to-fall progression in the northwestern Sargasso Sea to investigate the relationship between upper ocean stratification and phytoplankton nitrogen (N) uptake. Euphotic zone particles collected in July, October, and December were sorted by flow cytometry, and the 15N/14N of separated prokaryotic and eukaryotic phytoplankton was analyzed. The 15N/14N of Prochlorococcus and Synechococcus was always low, indicating uniform reliance on recycled N. In July and in two fall profiles, the 15N/14N of eukaryotic phytoplankton was high, reflecting consumption of subsurface nitrate. In three other fall profiles, eukaryotic 15N/14N was similar to prokaryote 15N/14N, suggesting a shift toward more complete reliance on recycled N. The progressive deepening of the mixed layer from summer to fall, although reducing the surface-to-deep density contrast, increases the density difference of the euphotic zone as a whole from underlying nutrient-rich waters, which may play a role in the observed decline in euphotic zone nitrate supply into the fall. The apparent summertime nitrate supply to the euphotic zone, when the mixed layer is shallowest, may help to explain the surprisingly high export production of the subtropical and tropical ocean. [ABSTRACT FROM AUTHOR]

Ammonia oxidation rates and ammonia-oxidizer community structure were examined in a depth profile (20-2000 m) at the Bermuda Atlantic Time-Series Study site in December 2009. Ammonia oxidation rates, measured from trace additions of 15 (12-18 nmol L-1), ranged from undetectable at the surface and 2000 m to 2.0 ± 0.1 nmol L-1 d-1 at 120 m, the depth of the primary nitrite maximum (PNM). Nitrification was not detectable in the photic zone in December, perhaps in part due to the density structure of the upper water column at this time. Ammonium oxidation rates varied with ammonium concentration and yielded an estimate for the half-saturation constant of 65 ± 41 nmol L-1 for the assemblage at 100 m. This value is similar to that reported for the cultivated marine ammonia-oxidizing archeon Nitrosopumilus maritimus (134 nmol L-1), confirming the high affinity for ammonium of the in situ community in the Sargasso Sea. Ammonia-oxidizing archaeal (AOA) amoA gene copy numbers were two orders of magnitude higher than ammonia-oxidizing bacterial (AOB) amoA gene copy numbers at the PNM depth, suggesting that AOA were responsible for most of the ammonium oxidation, which was in turn responsible for the formation of the PNM. AOB abundance exceeded AOA at depths below 140 m, where both groups were much less abundant. Application of an AOA amoA functional gene microarray showed a diverse and even community distribution. A single archetype (AOA24, representing sequences originally from a coral reef) had the highest fluorescence ratio at depths of 0-1000 m. [ABSTRACT FROM AUTHOR]

Summary Ammonia-oxidizing bacteria (AOB) and archaea (AOA) play a vital role in bridging the input of fixed nitrogen, through N-fixation and remineralization, to its loss by denitrification and anammox. Yet the major environmental factors determining AOB and AOA population dynamics are little understood, despite both groups having a wide environmental distribution. This study examined the relative abundance of both groups of ammonia-oxidizing organisms (AOO) and the diversity of AOA across large-scale gradients in temperature, salinity and substrate concentration and dissolved oxygen. The relative abundance of AOB and AOA varied across environments, with AOB dominating in the freshwater region of the Chesapeake Bay and AOA more abundant in the water column of the coastal and open ocean. The highest abundance of the AOA amoA gene was recorded in the oxygen minimum zones (OMZs) of the Eastern Tropical South Pacific (ETSP) and the Arabian Sea (AS). The ratio of AOA : AOB varied from 0.7 in the Chesapeake Bay to 1600 in the Sargasso Sea. Relative abundance of both groups strongly correlated with ammonium concentrations. AOA diversity, as determined by phylogenetic analysis of clone library sequences and archetype analysis from a functional gene DNA microarray, detected broad phylogenetic differences across the study sites. However, phylogenetic diversity within physicochemically congruent stations was more similar than would be expected by chance. This suggests that the prevailing geochemistry, rather than localized dispersal, is the major driving factor determining OTU distribution. [ABSTRACT FROM AUTHOR]

Nitrification rates, as well as the relationships between rates and ammonia oxidizer abundance (both archaeal and bacterial), were investigated in the Arabian Sea. Ammonia oxidation rates were measured directly using 15N-NH+4 stable isotope additions in gas-impermeable, trace metal clean trilaminate bags (500 mL) at in situ temperature. Tracer incubations were performed at three stations at depths above, below, and within the oxycline of the open-ocean oxygen minimum zone (OMZ). Ammonia oxidation rates were similar to previous open-ocean measurements, ranging from undetectable to 21.6 ± 0.1 nmol L-1 d-1. The highest rates at each station occurred at the primary nitrite maximum (above the OMZ), and rates were very low at depths greater than 900 m. The abundances of both ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB) were estimated using the amoA gene by quantitative polymerase chain reaction (qPCR). Both AOA and AOB amoA were detected above, within, and below the OMZ, although the AOA were always more abundant than the AOB, by a factor of 35-216. Nitrification rates were not directly correlated to AOA or AOB amoA abundance. These rates offer new insight into the role of nitrification in the mesopelagic zone. The abundance of AOA amoA genes at 1000 m suggests that ~50% of the microbial biomass could be autotrophic. Additionally, the integrated nitrification rate at depth implies that nitrification could consume most of the ammonium produced by the flux of organic carbon in the mesopelagic zone. [ABSTRACT FROM AUTHOR]

Microbes exhibit remarkably high genetic diversity compared with plant and animal species. Many phylogenetically diverse but apparently functionally redundant microbial taxa are detectable within a cubic centimetre of mud or a millilitre of water, and the significance of this diversity, in terms of ecosystem function, has been difficult to understand. Thus it is not known whether temporal and spatial differences in microbial community composition are linked to particular environmental factors or might modulate ecosystem response to environmental change. Fifty-three water and sediment samples from upper and lower Chesapeake Bay were analysed in triplicate arrays to determine temporal and spatial patterns and relationships between ammonia-oxidizing bacterial (AOB) communities and environmental variables. Thirty-three water samples (three depths) collected during April, August and October, 2001-2004, from the oligohaline upper region of the Bay were analysed to investigate temporal patterns in archetype distribution. Using a combination of a non-weighted discrimination analysis and principal components analysis of community composition data obtained from functional gene microarrays, it was found that co-varying AOB assemblages reoccurred seasonally in concert with specific environmental conditions, potentially revealing patterns of niche differentiation. Among the most notable patterns were correlations of AOB archetypes with temperature, DON and ammonium concentrations. Different AOB archetypes were more prevalent at certain times of the year, e.g. some were more abundant every autumn and others every spring. This data set documents the successional return to an indigenous community following massive perturbation (hurricane induced flooding) as well as the seasonal reoccurrence of specific lineages, identified by key functional genes, associated with the biogeochemically important process nitrification. [ABSTRACT FROM AUTHOR]

The phylogeny and diversity of two key functional genes were investigated as the basis for improved understanding of the community structure of natural phytoplankton assemblages in marine environments. New partial NR (encoding eukaryotic assimilatory nitrate reductase) and rbcL (encoding LSU of RUBISCO) sequences from 10 cultured phytoplankton strains are reported. Phytoplankton community composition from Monterey Bay (MB), a coastal upwelling site on the California coast, and the Western English Channel (EC), a North Atlantic spring bloom environment, was elucidated based on NR and rbcL sequences. Diatoms were by far the most frequently detected group in both environments, consistent with their importance as a major bloom-forming group. Both NR and rbcL libraries contained sequences representing cosmopolitan types such as Emiliania huxleyi (Lohmann) W. W. Hay et H. P. Mohler, Phaeocystis, and Pseudo-nitzschia. The NR and rbcL libraries also contained sequences from other chromophytic algal groups and the Dinophyceae (alveolates). Sequences showing identity with key bloom-forming organisms including E. huxleyi, Phaeocystis pouchetii (Har.) Lagerh., Pseudo-nitzschia sp., and Thalassiosira sp. in the rbcL libraries confirm previous studies from these environments based on traditional approaches. Diversity/pattern analyses detected significant compositional differences among the libraries, which were consistent with patterns identified by phylogenetic analysis, but these patterns were not strongly correlated with obvious environmental variables such as temperature and nitrate concentration. Many new and divergent NR and rbcL sequences are reported, but the extent to which they represent unknown types cannot be determined until greater effort is made to sequence the existing culture collections. [ABSTRACT FROM AUTHOR]

A functional gene microarray was used to investigate denitrifier community composition and nitrite reductase ( nirS) gene expression in sediments along the estuarine gradient in Chesapeake Bay, USA. The nirS oligonucleotide probe set was designed to represent a sequence database containing 539 Chesapeake Bay clones, as well as sequences from many other environments. Greatest nirS diversity was detected at the freshwater station at the head of the bay and least diversity at the higher salinity station near the mouth of the Bay. The most common OTUs from the sequence database were detected on the array with high signal strength in most samples. One of the most abundant OTUs, CB2-S-138, was identified as dominant at the mid-bay site by both microarray and quantitative PCR assays, but it comprised a much smaller fraction of the assemblage in the north and south bay samples. cDNA (transcribed from total RNA extracts) targets were hybridized to the same array to compare the profiles of community composition at the DNA (relative abundance) and mRNA (gene expression) levels. Only the three dominant denitrifying groups (in terms of relative strength of DNA hybridization signal) were detected at the mRNA level. These results suggest that the most actively denitrifying groups are responsible for most nirS expression as well. [ABSTRACT FROM AUTHOR]

The relationship between environmental factors and functional gene diversity of ammonia-oxidizing bacteria (AOB) was investigated across a transect from the freshwater portions of the Chesapeake Bay and Choptank River out into the Sargasso Sea. Oligonucleotide probes (70-bp) designed to represent the diversity of ammonia monooxygenase ( amoA) genes from Chesapeake Bay clone libraries and cultivated AOB were used to construct a glass slide microarray. Hybridization patterns among the probes in 14 samples along the transect showed clear variations in amoA community composition. Probes representing uncultivated members of the Nitrosospira-like AOB dominated the probe signal, especially in the more marine samples. Of the cultivated species, only Nitrosospira briensis was detected at appreciable levels. Discrimination analysis of hybridization signals detected two guilds. Guild 1 was dominated by the marine Nitrosospira-like probe signal, and Guild 2′s largest contribution was from upper bay (freshwater) sediment probes. Principal components analysis showed that Guild 1 was positively correlated with salinity, temperature and chlorophyll a concentration, while Guild 2 was positively correlated with concentrations of oxygen, dissolved organic carbon, and particulate nitrogen and carbon, suggesting that different amoA sequences represent organisms that occupy different ecological niches within the estuarine/marine environment. The trend from most diversity of AOB in the upper estuary towards dominance of a single type in the polyhaline region of the Bay is consistent with the declining importance of AOB with increasing salinity, and with the idea that AO-Archaea are the more important ammonia oxidizers in the ocean. [ABSTRACT FROM AUTHOR]

The distribution of nitrogen-fixing microorganisms in the Chesapeake Bay was investigated using fingerprints from a nifH microarray comprised of 706 60-mer oligonucleotide nifH probes representing cultivated organisms and environmental clones from different nifH clusters. Diverse nifH targets, amplified from samples using degenerate nifH primers, were detected in water column and sediment samples collected in April and October, 2001–2002. Total nifH richness and diversity (Simpson's and Shannon indices) were highest at the most riverine, oligohaline North Bay station. In most samples, the highest diversity was in nifH Cluster 3, which includes many anaerobes, while Cluster 1 (α-, β- γ- Proteobacteria, Cyanobacteria) targets had the greatest microarray signal intensities. In a multidimensional scaling analysis, deep water communities from April and October were similar within each of the sampling sites, while the surface communities had more variability. Diazotroph communities in the water column in the North Bay were distinct from the Mid- and South Bay communities, and there was a gradual change in sediment diazotroph assemblages from the North to the South Bay. Diazotrophic assemblages from the majority of the water column samples from the Mid- and South Bay clustered with the sediment assemblage in Mid-Bay. Dissolved inorganic nitrogen, salinity, dissolved organic carbon and dissolved organic phosphorus had a significant relationship with the diazotrophic bacterioplankton community. Higher diversity in the freshwater end of the system may reflect variability in disturbance rates and environmental conditions such as forms and concentrations of organic matter, nutrients and oxygen. [ABSTRACT FROM AUTHOR]

Nitrate transporter systems initiate nitrate uptake in photoautotrophic organisms including marine phytoplankton. The functional genes encoding high-affinity nitrate transporters ( Nrt2) in marine phytoplankton were investigated in this study. Direct PCR amplification with newly developed primers was used to detect Nrt2 genes in cultured marine phytoplankton species: Dunaliella tertiolecta Butcher (Chlorophyceae); Emiliania huxleyi (Lohmann) W. H. Hay et H. Mohler (Haptophyceae); and four diatoms, including Thalassiosira weissflogii (Grunow) G. A. Fryxell et Hasle, Skeletonema costatum (Grev.) Cleve, Ditylum brightwellii (T. West) Grunow, and Chaetoceros muelleri Lemmerm. (Bacillariophyceae). Phylogenetic analysis of the detected Nrt2 genes identified three distinct clusters grouped by each class of phytoplankton species. Transcriptional abundance (mRNA) of Nrt2 genes in D. tertiolecta, E. huxleyi, T. weissflogii, and C. muelleri were measured using quantitative reverse transcriptase-PCR assays under various nitrogen conditions. The diatoms and E. huxleyi induced Nrt2 gene transcripts both in the presence of nitrate and under nitrogen (N) starvation, while the Nrt2 transcript in D. tertiolecta was induced only when nitrate was present. Chaetoceros muelleri exhibited the highest transcript induction of the Nrt2 gene. The transcriptional abundance of the Nrt2 genes in C. muelleri was further examined with short-term nitrate incubation of cells grown under N-sufficient or N-starved conditions. The maximum induction of Nrt2 gene transcripts was observed within 1 h after nitrate was added. Thus, we report differential induction and regulation of Nrt2 genes in the selected phytoplankton species to varying conditions of external N. [ABSTRACT FROM AUTHOR]

The complete assimilatory nitrate reductase (NR) gene from the pennate diatomPhaeodactylum triconutumBohlin was sequenced from cDNA and compared with NR sequences from fungi, green algae, vascular plants, and the recently sequenced genome of the centric diatomThalassiosira pseudonanaHasle and Heimdal CCMP1335. In all the major eukaryotic nitrate reductase (Euk-NR) functional domains, diatom NR gene sequences are generally 50%–60% identical to plant and alga sequences at the amino acid level. In the less conserved N-terminal, hinge 1, and hinge 2 regions, homology to other NR sequences is weak, generally<30%. Two PCR primer sets capable of amplifying Euk-NR from plants, algae, and diatoms were designed. One primer set was used to amplify a 750-base pair (bp) NR fragment from the cDNA of five additional diatom strains. The PCR amplicon spans part of the well-conserved dimer interface region, the more variable hinge 1 region, and part of the conserved cytochromebheme binding region. The second primer set, targeted to the dimer region, was used to amplify an approximately 400-bp fragment of the NR gene from DNA samples collected in Monterey Bay, California and in central New Jersey inner continental shelf (LEO-15 site) waters. Only diatom-like NR sequences were recovered from Monterey Bay samples, whereas LEO-15 samples yielded NR sequences from a range of photosynthetic eukaryotes. The prospect of using DNA- and RNA-based methods to target the NR genes of diatoms specifically is a promising approach for future physiological and ecological experiments. [ABSTRACT FROM AUTHOR]

Nitrate is used as a nutrient for growth by many organisms through the nitrate assimilation pathway. The key enzyme involved in nitrate assimilation is nitrate reductase (NR). Transcriptional regulation of NR genes has been studied in various phototrophic eukaryotes from freshwater or terrestrial habitats. Here, we describe the first NR gene from a marine phytoplankton, the green alga Dunaliella tertiolecta Butcher. Its sequence is very similar to that of the other green algae, but its intron structure and transcriptional regulation differ significantly. A partial genomic NR gene fragment was cloned using direct PCR amplification with degenerate primers, and rapid amplification of cDNA ends was performed to obtain a complete NR gene from a cDNA of D. tertiolecta. The complete NR gene of D. tertiolecta is 2.7 kb in size and is similar at the amino acid level to the NRs from freshwater green algae Chlorella vulgaris (50.6%), Chlamydomonas reinhardtii (58.4%), and Volvox carteri (59.4%). Transcriptional regulation of D. tertiolecta NR was examined in response to different nitrogen sources and during the diel cycle. Dunaliella tertiolecta NR transcript was induced by nitrate and repressed by ammonium. In addition, transcription was not induced under nitrogen depletion. Induction of NR transcript responded to the diel cycle, and transcript levels were maximized within an hour after the beginning of the light period. The induction and repression of NR transcript were much slower than reported in freshwater green algae, which may be related to the size of internal nitrogen pools. [ABSTRACT FROM AUTHOR]

Chesapeake Bay, the largest estuary in North America, encompasses a wide range of nutrient loading and trophic levels from the rivers and upper Bay to the sea, providing an ideal natural environment in which to explore relationships between functional diversity, physical/chemical complexity and ecosystem function (e.g. nitrification). In this study, amoA gene fragments (encoding subunit A of the key nitrification enzyme, ammonia monooxygenase) were PCR-amplified from DNA extracted from sediment cores collected at five stations spanning gradients of salinity, ammonium, nitrate, oxygen and organic carbon along the Bay and Choptank River, a subestuary of the Bay. Phylogenetic analysis of ∼30 amoA clones from each station revealed extensive diversity within the β- Proteobacteria group of ammonia-oxidizing bacteria (AOB), with the vast majority of sequences falling into coherent phylogenetic clusters distinct from sequences of cultivated AOB. Over 70% of the clones fell into two major phylogenetic clusters that appear to represent novel groups of Nitrosomonas-like and Nitrosospira-like amoA sequences that may be specific to estuarine and marine environments. Rarefaction analysis, estimators of genetic variation and dissimilarity indices all revealed differences in the relative amoA-based diversity and/or richness among most of the stations, with the highest diversity at the North Bay station and the lowest at the mesohaline stations. Although salinity appears to play a role, no single physical or chemical parameter entirely explains the pattern of diversity along the estuary, suggesting that a complex combination of environmental factors may shape the overall level of AOB diversity in this dynamic environment. [ABSTRACT FROM AUTHOR]

Examines the accumulation of nitrogen oxides in copper-limited cultures of denitrifying bacteria. Cultivation of denitrifying bacteria under trace metal lean conditions; Growth under copper-replete conditions; Effect of copper concentrations on nitrate-dependent growth of Pseudomonas stutzeri and Pseudomonas denitrificans.