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Many estuaries experience eutrophication, deoxygenation and warming, with potential impacts on greenhouse gas emissions. However, the response of N₂O production to these changes is poorly constrained. Here we applied nitrogen isotope tracer incubations to measure N₂O production under experimentally manipulated changes in oxygen and temperature in the Chesapeake Bay—the largest estuary in the United States. N₂O production more than doubled from nitrification and increased exponentially from denitrification when O₂was decreased from >20 to <5 micromolar. Raising temperature from 15° to 35°C increased N₂O production 2- to 10-fold. Developing a biogeochemical model by incorporating these responses, N₂O emissions from the Chesapeake Bay were estimated to decrease from 157 to 140 Mg N year⁻¹from 1986 to 2016 and further to 124 Mg N year⁻¹in 2050. Although deoxygenation and warming stimulate N₂O production, the modeled decrease in N₂O emissions, attributed to decreased nutrient inputs, indicates the importance of nutrient management in curbing greenhouse gas emissions, potentially mitigating climate change. 

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 genenosZ. Organisms that containnosZare the only known biological sinks of N₂O and are found in diverse genera and a wide range of environments. The two clades ofnosZ(Clade I and II) contain great diversity, making it challenging to study the population structure and distribution ofnosZcontaining organisms in the environment. A database of over 11,000nosZsequences was compiled from NCBI (representing diverse aquatic environments) and unpublished sequences and metagenomes (primarily from oxygen minimum zones, OMZs, where N₂O levels are often elevated).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 InosZgenes in the compilation, Clade II is more diverse phylogenetically and has a wider distribution across environmental sources. On the other hand, Clade InosZgenes 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 thenosZgene displays regional and environmental separation. This study expands the known diversity ofnosZgenes and provides a clearer picture of how the clades and phylogeny ofnosZorganisms are distributed across diverse environments. 

Abstract 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. 

Abstract 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. 

Abstract 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. 

Abstract 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 Nitrous oxide (N 2 O) is important to the global radiative budget of the atmosphere and contributes to the depletion of stratospheric ozone. Globally the ocean represents a large net flux of N 2 O to the atmosphere but the direction of this flux varies regionally. Our understanding of N 2 O production and consumption processes in the ocean remains incomplete. Traditional understanding tells us that anaerobic denitrification, the reduction of NO 3 − to N 2 with N 2 O as an intermediate step, is the sole biological means of reducing N 2 O, a process known to occur in anoxic environments only. Here we present experimental evidence of N 2 O removal under fully oxygenated conditions, coupled with observations of bacterial communities with novel, atypical gene sequences for N 2 O reduction. The focus of this work was on the high latitude Atlantic Ocean where we show bacterial consumption sufficient to account for oceanic N 2 O depletion and the occurrence of regional sinks for atmospheric N 2 O. 

Abstract The ocean is a net source of N 2 O, a potent greenhouse gas and ozone-depleting agent. However, the removal of N 2 O via microbial N 2 O consumption is poorly constrained and rate measurements have been restricted to anoxic waters. Here we expand N 2 O 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, O 2 tolerance, and community composition of N 2 O-consuming microbes in oxic waters differ from those in the underlying anoxic layers. Kinetic parameters determined here are used to model in situ N 2 O production and consumption rates. Estimated in situ rates differ from measured rates, confirming the necessity to consider kinetics when predicting N 2 O cycling. Microbes from the oxic layer consume N 2 O 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 N 2 O consumption likely takes place above the oxygen deficient zone (ODZ). Thus, the dynamic layer with steep O 2 and N 2 O gradients right above the ODZ is a previously ignored potential gatekeeper of N 2 O and should be accounted for in the marine N 2 O budget. 

Nitrous oxide (N 2 O) is a potent greenhouse gas and an ozone destroying substance. Yet, clear step-by-step protocols to measure N 2 O transformation rates in freshwater and marine environments are still lacking, challenging inter-comparability efforts. Here we present detailed protocols currently used by leading experts in the field to measure water-column N 2 O production and consumption rates in both marine and other aquatic environments. We present example 15 N-tracer incubation experiments in marine environments as well as templates to calculate both N 2 O production and consumption rates. We discuss important considerations and recommendations regarding (1) precautions to prevent oxygen (O 2 ) contamination during low-oxygen and anoxic incubations, (2) preferred bottles and stoppers, (3) procedures for 15 N-tracer addition, and (4) the choice of a fixative. We finally discuss data reporting and archiving. We expect these protocols will make 15 N-labeled N 2 O transformation rate measurements more accessible to the wider community and facilitate future inter-comparison between different laboratories.