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In the current era of rapid climate change, accurate characterization of climate-relevant gas dynamics-namely production, consumption, and net emissions-is required for all biomes, especially those ecosystems most susceptible to the impact of change. Marine environments include regions that act as net sources or sinks for numerous climateactive trace gases including methane (CH4) and nitrous oxide (N2O). The temporal and spatial distributions of CH4 and N2O are controlled by the interaction of complex biogeochemical and physical processes. To evaluate and quantify how these mechanisms affect marine CH4 and N2O cycling requires a combination of traditional scientific disciplines including oceanography, microbiology, and numerical modeling. Fundamental to these efforts is ensuring that the datasets produced by independent scientists are comparable and interoperable. Equally critical is transparent communication within the research community about the technical improvements required to increase our collective understanding of marine CH4 and N2O. A workshop sponsored by Ocean Carbon and Biogeochemistry (OCB) was organized to enhance dialogue and collaborations pertaining to marine CH4 and N2O. Here, we summarize the outcomes from the workshop to describe the challenges and opportunities for near-future CH4 and N2O research in the marine environment.

Understanding the interactions between microbial communities and their environment sufficiently to predict diversity on the basis of physicochemical parameters is a fundamental pursuit of microbial ecology that still eludes us. However, modeling microbial communities is problematic, because (i) communities are complex, (ii) most descriptions are qualitative, and (iii) quantitative understanding of the way communities interact with their surroundings remains incomplete. One approach to overcoming such complications is the integration of partial qualitative and quantitative descriptions into more complex networks. Here we outline the development of a probabilistic framework, based on Event Transition Graph (ETG) theory, to predict microbial community structure across observed chemical data. Using reverse engineering, we derive probabilities from the ETG that accurately represent observations from experiments and predict putative constraints on communities within dynamic environments. These predictions can feedback into the future development of field experiments by emphasizing the most important functional reactions, and associated microbial strains, required to characterize microbial ecosystems.

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]

Nitrification, the stepwise oxidation of ammonia to nitrate via nitrite, is a key process in the marine nitrogen cycle. Reported nitrite oxidation rates frequently exceed ammonia oxidation rates below the euphotic zone, raising the fundamental question of whether the two steps are balanced and if alternative sources contribute to nitrite production in the dark ocean. Here we present vertically resolved profiles of ammonia, urea, and nitrite oxidation rates and their kinetic traits in the oligotrophic Subtropical North Pacific. Our results show active urea‐derived nitrogen oxidation (urea‐N oxidation) in the presence of experimental ammonium amendment, suggesting direct urea utilization. The depth‐integrated rates of urea‐N oxidation and ammonia oxidation are comparable, demonstrating that urea‐N oxidation is a significant source of nitrite. The additional nitrite from urea‐N oxidation helps to balance the two steps of nitrification in our study region. Nitrifiers exhibit high affinity for their substrates, and the apparent half‐saturation constants for ammonia and nitrite oxidation decrease with depth. The apparent half‐saturation constant for urea‐N oxidation is higher than that for ammonia oxidation and shows no clear vertical trend. Such kinetic traits may account for the relatively higher urea concentration than ammonium concentration in the ocean's interior. Moreover, a compilation of our results and reported data shows a trend of increased urea‐N oxidation relative to ammonia oxidation from the eutrophic coastal zone to the oligotrophic open ocean. This trend reveals a substrate‐dependent biogeographic distribution of urea‐N oxidation across marine environments and provides new information on the balance and flux of the marine nitrification process. Key Points: Active urea‐derived nitrogen oxidation in the presence of added ammonium suggests direct urea utilization by marine ammonia oxidizersSubstrate affinity regulates the vertical distribution of ammonia, urea, and nitrite in the ocean's interiorNitrite production from urea is comparable to that from ammonia oxidation in the energy‐starved mesopelagic ocean [ABSTRACT FROM AUTHOR]

Understanding the interactions between microbial communities and their environment sufficiently to predict diversity on the basis of physicochemical parameters is a fundamental pursuit of microbial ecology that still eludes us. However, modeling microbial communities is problematic, because (i) communities are complex, (ii) most descriptions are qualitative, and (iii) quantitative understanding of the way communities interact with their surroundings remains incomplete. One approach to overcoming such complications is the integration of partial qualitative and quantitative descriptions into more complex networks. Here we outline the development of a probabilistic framework, based on Event Transition Graph (ETG) theory, to predict microbial community structure across observed chemical data. Using reverse engineering, we derive probabilities from the ETG that accurately represent observations from experiments and predict putative constraints on communities within dynamic environments. These predictions can feedback into the future development of field experiments by emphasizing the most important functional reactions, and associated microbial strains, required to characterize microbial ecosystems.

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]

Nitrous oxide (N 2 O) is a potent greenhouse gas and ozone depletion agent, with a significant natural source from marine oxygen-deficient zones (ODZs). Open questions remain, however, about the microbial processes responsible for this N 2 O production, especially hybrid N 2 O production when ammonia-oxidizing archaea are present. Using 15 N-labeled tracer incubations, we measured the rates of N 2 O production from ammonium (NH 4+), nitrite (NO 2-), and nitrate (NO 3-) in the eastern tropical North Pacific ODZ and the isotopic labeling of the central (α) and terminal (β) nitrogen (N) atoms of the N 2 O molecule. We observed production of both doubly and singly labeled N 2 O from each tracer, with the highest rates of labeled N 2 O production at the same depths as the near-surface N 2 O concentration maximum. At most stations and depths, the production of 45 N 2 O α and 45 N 2 O β were statistically indistinguishable, but at a few depths there were significant differences in the labeling of the two nitrogen atoms in the N 2 O molecule. Implementing the rates of labeled N 2 O production in a time-dependent numerical model, we found that N 2 O production from NO 3- dominated at most stations and depths, with rates as high as 1600 ± 200 pM N 2 O d -1. Hybrid N 2 O production, one of the mechanisms by which ammonia-oxidizing archaea produce N 2 O, had rates as high as 230 ± 80 pM N 2 O d -1 that peaked in both the near-surface and deep N 2 O concentration maxima. Based on the equal production of 45 N 2 O α and 45 N 2 O β in the majority of our experiments, we infer that hybrid N 2 O production likely has a consistent site preference, despite drawing from two distinct substrate pools. We also found that the rates and yields of hybrid N 2 O production were enhanced at low dissolved oxygen concentrations ([O 2 ]), with hybrid N 2 O yields as high as 20 % at depths where [O 2 ] was below detection (880 nM) but nitrification was still active. Finally, we identified a few incubations with [O 2 ] up to 20 µ M where N 2 O production from NO 3- was still active. A relatively high O 2 tolerance for N 2 O production via denitrification has implications for the feedbacks between marine deoxygenation and greenhouse gas cycling. [ABSTRACT FROM AUTHOR]

Nitrous oxide (N 2 O), a potent greenhouse gas and ozone-destroying agent, is produced during nitrogen transformations in both natural and human-constructed environments. Wastewater treatment plants (WWTPs) produce and emit N 2 O into the atmosphere during the nitrogen removal process. However, the impact of WWTPs on N 2 O emissions in downstream aquatic systems remains poorly constrained. By measuring N 2 O concentrations at a monthly resolution over a year in the Potomac River estuary, a tributary of the Chesapeake Bay in the eastern United States, we found a strong seasonal variation in N 2 O concentrations and fluxes: N 2 O concentrations were larger in fall and winter, but the flux was larger in summer and fall. Observations at multiple stations across the Potomac River estuary revealed hotspots of N 2 O emissions downstream of WWTPs. N 2 O concentrations were higher at stations downstream of WWTPs compared to other stations (median: 21.2 nM vs. 16.2 nM) despite the similar concentration of dissolved inorganic nitrogen, suggesting the direct discharge of N 2 O from WWTPs into the aquatic system or a higher N 2 O production yield in waters influenced by WWTPs. Meta-analysis of N 2 O measurements associated with WWTPs globally revealed variable influence of WWTPs on downstream N 2 O concentrations and emissions. Since wastewater production has increased substantially with the growing population and is projected to continue to rise, accurately accounting for N 2 O emissions downstream of WWTPs is important for constraining and predicting future global N 2 O emissions. Efficient N 2 O removal, in addition to dissolved nitrogen removal, should be an essential part of water quality control in WWTPs. [ABSTRACT FROM AUTHOR]

Metagenomic approaches have revealed unprecedented genetic diversity within microbial communities across vast expanses of the world's oceans. Linking this genetic diversity with key metabolic and cellular activities of microbial assemblages is a fundamental challenge. Here we report on a collaborative effort to design MicroTOOLs (Microbiological Targets for Ocean Observing Laboratories), a high-density oligonucleotide microarray that targets functional genes of diverse taxa in pelagic and coastal marine microbial communities. MicroTOOLs integrates nucleotide sequence information from disparate data types: genomes, PCR-amplicons, metagenomes, and metatranscriptomes. It targets 19 400 unique sequences over 145 different genes that are relevant to stress responses and microbial metabolism across the three domains of life and viruses. MicroTOOLs was used in a proof-of-concept experiment that compared the functional responses of microbial communities following Fe and P enrichments of surface water samples from the North Pacific Subtropical Gyre. We detected transcription of 68% of the gene targets across major taxonomic groups, and the pattern of transcription indicated relief from Fe limitation and transition to N limitation in some taxa. Prochlorococcus (eHLI), Synechococcus (sub-cluster 5.3) and Alphaproteobacteria SAR11 clade (HIMB59) showed the strongest responses to the Fe enrichment. In addition, members of uncharacterized lineages also responded. The MicroTOOLs microarray provides a robust tool for comprehensive characterization of major functional groups of microbes in the open ocean, and the design can be easily amended for specific environments and research questions.

Nitrous oxide (N2O), a potent greenhouse gas and ozone-destroying agent, is produced during nitrogen transformations in both natural and human-constructed environments. Wastewater treatment plants (WWTPs) produce and emit N2O into the atmosphere during the nitrogen removal process. However, the impact of WWTPs on N2O emissions in downstream aquatic systems remains poorly constrained. By measuring N2O concentrations at a monthly resolution over a year in the Potomac River Estuary, a tributary of Chesapeake Bay in the eastern United States, we found a strong seasonal variation in N2O concentrations and fluxes: N2O concentrations were larger in fall and winter but the flux was larger in summer and fall. Observations at multiple stations across the Potomac River Estuary revealed hotspots of N2O emissions downstream of WWTPs. N2O concentrations were higher at stations downstream of WWTPs compared to other stations (median: 21.2 nM vs 16.2 nM) despite the similar concentration of dissolved inorganic nitrogen, suggesting the direct discharge of N2O from WWTPs into the aquatic system or a higher N2O production yield in waters influenced by WWTPs. Since wastewater production has increased substantially with the growing population and is projected to continue to rise, accurately accounting for N2O emissions downstream of the WWTPs would better constrain the global N2O emissions. Efficient N2O removal, in addition to dissolved nitrogen removal, should be an essential part of water quality control in WWTPs. [ABSTRACT FROM AUTHOR]

Nitrous oxide (N2O) is a potent greenhouse gas and ozone depletion agent, with a significant natural source from marine oxygen deficient zones (ODZs). Open questions remain, however, about the microbial processes responsible for this N2O production, especially hybrid N2O production when ammonia-oxidizing archaea are present. Using 15N-labeled tracer incubations, we measured the rates of N2O production from ammonium (NH4+), nitrite (NO2-), and nitrate (NO3-) in the Eastern Tropical North Pacific ODZ, as well as the isotopic labeling of the central (α) and terminal (β) nitrogen atoms of the N2O molecule. We observed production of both doubly- and singly labeled N2O from each tracer, with the highest rates of labeled N2O production at the same depths as the near-surface N2O concentration maximum. At most stations and depths, the production of 45N2Oα and 45N2Oβ were statistically indistinguishable, but at a few depths, there were significant differences in the labelling of the two nitrogen atoms in the N2o molecule. Implementing the rates of labeled N2O production in a forward-running model, we found that N2O production from NO3- dominated at most stations and depths, with rates as high as 1.6±0.2 nM N2O/day. Hybrid N2O production, one of the mechanisms by which ammonia-oxidizing archaea produce N2O, had rates as high as 0.23±0.08 nM N2O/day that peaked in both the near-surface and deep N2O concentration maxima. We inferred from the 45N2Oα and 45N2Oβ data that hybrid N2O production by ammonia-oxidizing archaea may have a variable site preference that depends on the 15N content of each substrate. We also found that the rates and yields of hybrid N2O production exhibited a clear [O2] inhibition curve, with the hybrid N2O yields as high as 20 % at depths where dissolved [O2] was 0 µM but nitrification was still active. Finally, we identified a few incubations with dissolved [O2] up to 20 µM where N2O production from NO3- was still active. A relatively high O2 tolerance for N2O production via denitrification has implications for the feedbacks between marine deoxygenation and greenhouse gas cycling. [ABSTRACT FROM AUTHOR]

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.Incomplete denitrification associated with the micro-niche of marine particles, instead of nitrification as previously assumed, is a major source of N2O in the turbid coastal waters, even though the water is well-oxygenated. [ABSTRACT FROM AUTHOR]