Pathways of N2O production by marine ammonia-oxidizing archaea determined from dual-isotope labeling.

The ocean is a net source of the greenhouse gas and ozone-depleting substance, nitrous oxide (N2O), to the atmosphere. Most of that N2O is produced as a trace side product during ammonia oxidation, primarily by ammonia-oxidizing archaea (AOA), which numerically dominate the ammonia-oxidizing community in most marine environments. The pathways to N2O production and their kinetics, however, are not completely understood. Here, we use 15N and 18O isotopes to determine the kinetics of N2O production and trace the source of nitrogen (N) and oxygen (O) atoms in N2O produced by a model marine AOA species, Nitrosopumilus maritimus. We find that during ammonia oxidation, the apparent half saturation constants of nitrite and N2O production are comparable, suggesting that both processes are enzymatically controlled and tightly coupled at low ammonia concentrations. The constituent atoms in N2O are derived from ammonia, nitrite, O2, and H2O via multiple pathways. Ammonia is the primary source of N atoms in N2O, but its contribution varies with ammonia to nitrite ratio. The ratio of 45N2O to 46N2O (i.e., single or double labeled N) varies with substrate ratio, leading to widely varying isotopic signatures in the N2O pool. O2 is the primary source for O atoms. In addition to the previously demonstrated hybrid formation pathway, we found a substantial contribution by hydroxylamine oxidation, while nitrite reduction is an insignificant source of N2O. Our study highlights the power of dual 15N-18O isotope labeling to disentangle N2O production pathways in microbes, with implications for interpretation of pathways and regulation of marine N2O sources.

Evaluation of Genomic Sequence-Based Growth Rate Methods for Synchronized Synechococcus Cultures.

Standard methods for calculating microbial growth rates (µ) through the use of proxies, such as in situ fluorescence, cell cycle, or cell counts, are critical for determining the magnitude of the role bacteria play in marine carbon (C) and nitrogen (N) cycles. Taxon-specific growth rates in mixed assemblages would be useful for attributing biogeochemical processes to individual species and understanding niche differentiation among related clades, such as found in Synechococcus and Prochlorococcus. We tested three novel DNA sequencing-based methods (iRep, bPTR, and GRiD) for evaluating the growth of light-synchronized Synechococcus cultures under different light intensities and temperatures. In vivo fluorescence and cell cycle analysis were used to obtain standard estimates of growth rate for comparison with those of the sequence-based methods (SBM). None of the SBM values were correlated with growth rates calculated by standard techniques despite the fact that all three SBM were correlated with the percentage of cells in S phase (DNA replication) over the diel cycle. Inaccuracy in determining the time of maximum DNA replication is unlikely to account entirely for the absence of a relationship between SBM and growth rate, but the fact that most microbes in the surface ocean exhibit some degree of diel cyclicity is a caution for application of these methods. SBM correlate with DNA replication but cannot be interpreted quantitatively in terms of growth rate. IMPORTANCE Small but abundant, cyanobacterial strains such as the photosynthetic Synechococcus spp. are important because they contribute significantly to primary productivity in the ocean. These bacteria generate oxygen and provide biologically available carbon, which is essential for organisms at higher trophic levels. The small size and diversity of natural microbial assemblages mean that taxon-specific activities (e.g., growth rate) are difficult to obtain in the field. It has been suggested that sequence-based methods (SBM) may be able to solve this problem. We find, however, that SBM can detect DNA replication and are correlated with phases of the cell cycle but cannot be interpreted in terms of absolute growth rate for Synechococcus cultures growing under a day-night cycle, like that experienced in the ocean.

Global Nitrogen Cycle: Critical Enzymes, Organisms, and Processes for Nitrogen Budgets and Dynamics.

Nitrogen (N) is used in many of life's fundamental biomolecules, and it is also a participant in environmental redox chemistry. Biogeochemical processes control the amount and form of N available to organisms ("fixed" N). These interacting processes result in N acting as the proximate limiting nutrient in most surface environments. Here, we review the global biogeochemical cycle of N and its anthropogenic perturbation. We introduce important reservoirs and processes affecting N in the environment, focusing on the ocean, in which N cycling is more generalizable than in terrestrial systems, which are more heterogeneous. Particular attention is given to processes that create and destroy fixed N because these comprise the fixed N input/output budget, the most universal control on environmental N availability. We discuss preindustrial N budgets for terrestrial and marine systems and their modern-day alteration by N inputs from human activities. We summarize evidence indicating that the simultaneous roles of N as a required biomass constituent and an environmental redox intermediate lead to stabilizing feedbacks that tend to blunt the impact of N cycle perturbations at larger spatiotemporal scales, particularly in marine systems. As a result of these feedbacks, the anthropogenic "N problem" is distinct from the "carbon dioxide problem" in being more local and less global, more immediate and less persistent.

Long-Term Fertilization Alters Nitrous Oxide Cycling Dynamics in Salt Marsh Sediments.

Salt marsh sediments are known hotspots for nitrogen cycling, including the production and consumption of nitrous oxide (N2O), a potent greenhouse gas and ozone-depleting agent. Coastal eutrophication, particularly elevated nitrogen loading from the application of fertilizers, is accelerating nitrogen cycling processes in salt marsh sediments. Here, we examine the impact of long-term fertilization on nitrogen cycling processes with a focus on N2O dynamics in a New England salt marsh. By combining 15N-tracer experiments with numerical modeling, we found that both nitrification and denitrification contribute to net N2O production in fertilized sediments. Long-term fertilization increased the relative importance of nitrification to N2O production, likely a result of increased oxygen penetration from nutrient-induced increases in marsh elevation. Substrate utilization rates of key nitrogen cycling processes revealed links between functions and the corresponding microbial communities. Higher specific substrate utilization rates leading to N2O production from nitrification in fertilized sediments indicate a shift in the community composition of ammonia oxidizers, whereas the lack of change in specific substrate utilization of N2O production from denitrification under long-term fertilization suggests resilience of the denitrifying communities. Both are consistent with previous studies on the functional gene community composition in these experimental plots.

Organic Matter Loading Modifies the Microbial Community Responsible for Nitrogen Loss in Estuarine Sediments.

Coastal marine sediments, as locations of substantial fixed nitrogen loss, are very important to the nitrogen budget and to the primary productivity of the oceans. Coastal sediment systems are also highly dynamic and subject to periodic natural and anthropogenic organic substrate additions. The response to organic matter by the microbial community involved in nitrogen loss processes was evaluated using mesocosms of Chesapeake Bay sediments. Over the course of a 50-day incubation, rates of anammox and denitrification were measured weekly using (15)N tracer incubations, and samples were collected for genetic analysis. Rates of both nitrogen loss processes and gene abundances associated with them corresponded loosely, probably because heterogeneities in sediments obscured a clear relationship. The rates of denitrification were stimulated more, and the fraction of nitrogen loss attributed to anammox slightly reduced, by the higher organic matter addition. Furthermore, the large organic matter pulse drove a significant and rapid shift in the denitrifier community composition as determined using a nirS microarray, indicating that the diversity of these organisms plays an essential role in responding to anthropogenic inputs. We also suggest that the proportion of nitrogen loss due to anammox in these coastal estuarine sediments may be underestimated due to temporal dynamics as well as from methodological artifacts related to conventional sediment slurry incubation approaches.

Marine Oxygen-Deficient Zones Harbor Depauperate Denitrifying Communities Compared to Novel Genetic Diversity in Coastal Sediments.

Denitrification is a critically important biogeochemical pathway that removes fixed nitrogen from ecosystems and thus ultimately controls the rate of primary production in nitrogen-limited systems. We examined the community structure of bacteria containing the nirS gene, a signature gene in the denitrification pathway, from estuarine and salt marsh sediments and from the water column of two of the world's largest marine oxygen-deficient zones (ODZs). We generated over 125,000 nirS gene sequences, revealing a large degree of genetic diversity including 1,815 unique taxa, the vast majority of which formed clades that contain no cultured representatives. These results underscore how little we know about the genetic diversity of metabolisms underlying this critical biogeochemical pathway. Marine sediments yielded 1,776 unique taxa when clustered at 95 % sequence identity, and there was no single nirS denitrifier that was a competitive dominant; different samples had different highly abundant taxa. By contrast, there were only 39 unique taxa identified in samples from the two ODZs, and 99 % of the sequences belonged to 5 or fewer taxa. The ODZ samples were often dominated by nirS sequences that shared a 92 % sequence identity to a nirS found in the anaerobic ammonium-oxidizing (anammox) genus Scalindua. This sequence was abundant in both ODZs, accounting for 38 and 59 % of all sequences, but it was virtually absent in marine sediments. Our data indicate that ODZs are remarkably depauperate in nirS genes compared to the remarkable genetic richness found in coastal sediments.

Aquatic nitrous oxide reductase gene (nosZ) phylogeny and environmental distribution.

Nitrous oxide (N2O) is a potent greenhouse gas and a major cause of ozone depletion. One-third of atmospheric N2O originates in aquatic environments. Reduction of N2O to dinitrogen gas (N2) requires the nitrous oxide reductase enzyme, which is encoded by the gene nosZ. Organisms that contain nosZ are the only known biological sinks of N2O and are found in diverse genera and a wide range of environments. The two clades of nosZ (Clade I and II) contain great diversity, making it challenging to study the population structure and distribution of nosZ containing organisms in the environment. A database of over 11,000 nosZ sequences was compiled from NCBI (representing diverse aquatic environments) and unpublished sequences and metagenomes (primarily from oxygen minimum zones, OMZs, where N2O levels are often elevated). Sequences were clustered into archetypes based on DNA and amino acid sequence identity and their clade, phylogeny, and environmental source were determined. Further analysis of the source and environmental distribution of the sequences showed strong habitat separation between clades and phylogeny. Although there are more Clade I nosZ genes in the compilation, Clade II is more diverse phylogenetically and has a wider distribution across environmental sources. On the other hand, Clade I nosZ genes are predominately found within marine sediment and are primarily from the phylum Pseudonomonadota. The majority of the sequences analyzed from marine OMZs represented distinct phylotypes between different OMZs showing that the nosZ gene displays regional and environmental separation. This study expands the known diversity of nosZ genes and provides a clearer picture of how the clades and phylogeny of nosZ organisms are distributed across diverse environments.

Nitrite-oxidizing bacteria adapted to low-oxygen conditions dominate nitrite oxidation in marine oxygen minimum zones.

Nitrite is a central molecule in the nitrogen cycle because nitrite oxidation to nitrate (an aerobic process) retains fixed nitrogen in a system and its reduction to dinitrogen gas (anaerobic) reduces the fixed nitrogen inventory. Despite its acknowledged requirement for oxygen, nitrite oxidation is observed in oxygen-depleted layers of the ocean's oxygen minimum zones (OMZs), challenging the current understanding of OMZ nitrogen cycling. Previous attempts to determine whether nitrite-oxidizing bacteria in the anoxic layer differ from known nitrite oxidizers in the open ocean were limited by cultivation difficulties and sequencing depth. Here, we construct 31 draft genomes of nitrite-oxidizing bacteria from global OMZs. The distribution of nitrite oxidation rates, abundance and expression of nitrite oxidoreductase genes, and relative abundance of nitrite-oxidizing bacterial draft genomes from the same samples all show peaks in the core of the oxygen-depleted zone (ODZ) and are all highly correlated in depth profiles within the major ocean oxygen minimum zones. The ODZ nitrite oxidizers are not found in the Tara Oceans global dataset (the most complete oxic ocean dataset), and the major nitrite oxidizers found in the oxygenated ocean do not occur in ODZ waters. A pangenomic analysis shows the ODZ nitrite oxidizers have distinct gene clusters compared to oxic nitrite oxidizers and are microaerophilic. These findings all indicate the existence of nitrite oxidizers whose niche is oxygen-deficient seawater. Thus, specialist nitrite-oxidizing bacteria are responsible for fixed nitrogen retention in marine oxygen minimum zones, with implications for control of the ocean's fixed nitrogen inventory.

Age, metabolisms, and potential origin of dominant anammox bacteria in the global oxygen-deficient zones.

Anammox bacteria inhabiting oxygen-deficient zones (ODZs) are a major functional group mediating fixed nitrogen loss in the global ocean. However, many basic questions regarding the diversity, broad metabolisms, origin, and adaptive mechanisms of ODZ anammox bacteria remain unaddressed. Here we report two novel metagenome-assembled genomes of anammox bacteria affiliated with the Scalindua genus, which represent most, if not all, of the anammox bacteria in the global ODZs. Metagenomic read-recruiting and comparison with historical data show that they are ubiquitously present in all three major ODZs. Beyond the core anammox metabolism, both organisms contain cyanase, and the more dominant one encodes a urease, indicating most ODZ anammox bacteria can utilize cyanate and urea in addition to ammonium. Molecular clock analysis suggests that the evolutionary radiation of these bacteria into ODZs occurred no earlier than 310 million years ago, ~1 billion years after the emergence of the earliest modern-type ODZs. Different strains of the ODZ Scalindua species are also found in benthic sediments, and the first ODZ Scalindua is likely derived from the benthos. Compared to benthic strains of the same clade, ODZ Scalindua uniquely encodes genes for urea utilization but has lost genes related to growth arrest, flagellum synthesis, and chemotaxis, presumably for adaptation to thrive in the global ODZ waters. Our findings expand the known metabolisms and evolutionary history of the bacteria controlling the global nitrogen budget.

Ammonia-oxidizing bacteria and archaea exhibit differential nitrogen source preferences.

Ammonia-oxidizing microorganisms (AOM) contribute to one of the largest nitrogen fluxes in the global nitrogen budget. Four distinct lineages of AOM: ammonia-oxidizing archaea (AOA), beta- and gamma-proteobacterial ammonia-oxidizing bacteria (ß-AOB and γ-AOB) and complete ammonia oxidizers (comammox), are thought to compete for ammonia as their primary nitrogen substrate. In addition, many AOM species can utilize urea as an alternative energy and nitrogen source through hydrolysis to ammonia. How the coordination of ammonia and urea metabolism in AOM influences their ecology remains poorly understood. Here we use stable isotope tracing, kinetics and transcriptomics experiments to show that representatives of the AOM lineages employ distinct regulatory strategies for ammonia or urea utilization, thereby minimizing direct substrate competition. The tested AOA and comammox species preferentially used ammonia over urea, while ß-AOB favoured urea utilization, repressed ammonia transport in the presence of urea and showed higher affinity for urea than for ammonia. Characterized γ-AOB co-utilized both substrates. These results reveal contrasting niche adaptation and coexistence patterns among the major AOM lineages.

Controls on nitrogen loss processes in Chesapeake Bay sediments.

The flux of fixed nitrogen into the marine environment is increasing as a direct result of anthropogenic nitrogen loading, but the controls on the mechanisms responsible for the removal of this increased supply are not well constrained. The fate of fixed nitrogen via mineralization and nitrogen loss processes was investigated by simulating a settling event of organic matter (OM) in mesocosms containing Chesapeake Bay sediments. Microorganisms rapidly transformed the OM during the course of a seven week incubation ultimately leading to nitrogen loss via denitrification and anaerobic ammonium oxidation (anammox). The microbial community responded quickly to the OM amendment suggesting that estuarine sediments can buffer the natural system against sudden injections of organic material. Two different levels of organic matter amendment resulted in different magnitudes of ammonium and nitrite accumulation during the incubation, but both treatments exhibited the same overall sequence of dissolved inorganic nitrogen (DIN) accumulation and removal. An inverse least-squares analysis coupled to a Michaelis-Menten prognostic model was conducted to estimate rates of nitrogen transformations from the measured DIN concentrations. Whereas the rates were higher at higher OM, the percentage of nitrogen lost via anammox was constant at 44.3 ± 0.3%. The stoichiometry of organic matter and the allochthonous supply of ammonium determined the relative contribution of anammox and denitrification to overall nitrogen loss. Further, in situ thermodynamics based on measured concentrations suggested that the energy favorability of denitrification and anammox plays a role in determining the timing of these processes as OM remineralization progresses.

Partitioning of the denitrification pathway and other nitrite metabolisms within global oxygen deficient zones.

Oxygen deficient zones (ODZs) account for about 30% of total oceanic fixed nitrogen loss via processes including denitrification, a microbially mediated pathway proceeding stepwise from NO3- to N2. This process may be performed entirely by complete denitrifiers capable of all four enzymatic steps, but many organisms possess only partial denitrification pathways, either producing or consuming key intermediates such as the greenhouse gas N2O. Metagenomics and marker gene surveys have revealed a diversity of denitrification genes within ODZs, but whether these genes co-occur within complete or partial denitrifiers and the identities of denitrifying taxa remain open questions. We assemble genomes from metagenomes spanning the ETNP and Arabian Sea, and map these metagenome-assembled genomes (MAGs) to 56 metagenomes from all three major ODZs to reveal the predominance of partial denitrifiers, particularly single-step denitrifiers. We find niche differentiation among nitrogen-cycling organisms, with communities performing each nitrogen transformation distinct in taxonomic identity and motility traits. Our collection of 962 MAGs presents the largest collection of pelagic ODZ microorganisms and reveals a clearer picture of the nitrogen cycling community within this environment.

Particle-associated denitrification is the primary source of N2O in oxic coastal waters.

The heavily human-perturbed coastal oceans are hotspots of nitrous oxide (N2O) emission to the atmosphere. The processes underpinning the N2O flux, however, remain poorly understood, leading to large uncertainties in assessing global N2O budgets. Using a suite of nitrogen isotope labeling experiments, we show that multiple processes contribute to N2O production throughout the estuarine-coastal gradient, sustaining intensive N2O flux to the atmosphere. Unexpectedly, denitrification, rather than ammonia oxidation as previously assumed, constitutes the major source of N2O in well-oxygenated coastal waters. Size-fractionated manipulation experiments with gene analysis further reveal niche partitioning of ammonia oxidizers and denitrifiers across the particle size spectrum; denitrification dominated on large particles and ammonia oxidizers on small particles. Total N2O production rate increases with substrate and particle concentrations, suggesting a crucial interplay between nutrients and particles in controlling N2O production. The controlling factors identified here may help understand climate feedback mechanisms between human activity and coastal oceans.

Environmental factors determining ammonia-oxidizing organism distribution and diversity in marine environments.

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.

Seasonal and annual reoccurrence in betaproteobacterial ammonia-oxidizing bacterial population structure.

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.

Novel metagenome-assembled genomes involved in the nitrogen cycle from a Pacific oxygen minimum zone.

Oxygen minimum zones (OMZs) are unique marine regions where broad redox gradients stimulate biogeochemical cycles. Despite the important and unique role of OMZ microbes in these cycles, they are less characterized than microbes from the oxic ocean. Here we recovered 39 high- and medium-quality metagenome-assembled genomes (MAGs) from the Eastern Tropical South Pacific OMZ. More than half of these MAGs were not represented at the species level among 2631 MAGs from global marine datasets. OMZ MAGs were dominated by denitrifiers catalyzing nitrogen loss and especially MAGs with partial denitrification metabolism. A novel bacterial genome with nitrate-reducing potential could only be assigned to the phylum level. A Marine-Group II archaeon was found to be a versatile denitrifier, with the potential capability to respire multiple nitrogen compounds including N2O. The newly discovered denitrifying MAGs will improve our understanding of microbial adaptation strategies and the evolution of denitrification in the tree of life.

Microbial niche differentiation explains nitrite oxidation in marine oxygen minimum zones.

Nitrite is a pivotal component of the marine nitrogen cycle. The fate of nitrite determines the loss or retention of fixed nitrogen, an essential nutrient for all organisms. Loss occurs via anaerobic nitrite reduction to gases during denitrification and anammox, while retention occurs via nitrite oxidation to nitrate. Nitrite oxidation is usually represented in biogeochemical models by one kinetic parameter and one oxygen threshold, below which nitrite oxidation is set to zero. Here we find that the responses of nitrite oxidation to nitrite and oxygen concentrations vary along a redox gradient in a Pacific Ocean oxygen minimum zone, indicating niche differentiation of nitrite-oxidizing assemblages. Notably, we observe the full inhibition of nitrite oxidation by oxygen addition and nitrite oxidation coupled with nitrogen loss in the absence of oxygen consumption in samples collected from anoxic waters. Nitrite-oxidizing bacteria, including novel clades with high relative abundance in anoxic depths, were also detected in the same samples. Mechanisms corresponding to niche differentiation of nitrite-oxidizing bacteria across the redox gradient are considered. Implementing these mechanisms in biogeochemical models has a significant effect on the estimated fixed nitrogen budget.

Microbial N2O consumption in and above marine N2O production hotspots.

The ocean is a net source of N2O, a potent greenhouse gas and ozone-depleting agent. However, the removal of N2O via microbial N2O consumption is poorly constrained and rate measurements have been restricted to anoxic waters. Here we expand N2O consumption measurements from anoxic zones to the sharp oxygen gradient above them, and experimentally determine kinetic parameters in both oxic and anoxic seawater for the first time. We find that the substrate affinity, O2 tolerance, and community composition of N2O-consuming microbes in oxic waters differ from those in the underlying anoxic layers. Kinetic parameters determined here are used to model in situ N2O production and consumption rates. Estimated in situ rates differ from measured rates, confirming the necessity to consider kinetics when predicting N2O cycling. Microbes from the oxic layer consume N2O under anoxic conditions at a much faster rate than microbes from anoxic zones. These experimental results are in keeping with model results which indicate that N2O consumption likely takes place above the oxygen deficient zone (ODZ). Thus, the dynamic layer with steep O2 and N2O gradients right above the ODZ is a previously ignored potential gatekeeper of N2O and should be accounted for in the marine N2O budget.