Karen L. Casciotti’s research while affiliated with Stanford University and other places

Urea is hypothesized to be an important source of nitrogen and chemical energy to microorganisms in the deep sea; however, direct evidence for urea use below the epipelagic ocean is lacking. Here, we explore urea utilization from 50 to 4000 meters depth in the northeastern Pacific Ocean using metagenomics, nitrification rates, and single-cell stable-isotope-uptake measurements with nanoscale secondary ion mass spectrometry. We find that on average 25% of deep-sea cells assimilated urea-derived N (60% of detectably active cells), and that cell-specific nitrogen-incorporation rates from urea were higher than that from ammonium. Both urea concentrations and assimilation rates relative to ammonium generally increased below the euphotic zone. We detected ammonia- and urea-based nitrification at all depths at one of two sites analyzed, demonstrating their potential to support chemoautotrophy in the mesopelagic and bathypelagic regions. Using newly generated metagenomes we find that the ureC gene, encoding the catalytic subunit of urease, is found within 39% of deep-sea cells in this region, including the Nitrososphaeria (syn., Thaumarchaeota; likely for nitrification) as well as members of thirteen other phyla such as Proteobacteria, Verrucomicrobia, Plantomycetota, Nitrospinota, and Chloroflexota (likely for assimilation). Analysis of public metagenomes estimated ureC within 10–46% of deep-sea cells around the world, with higher prevalence below the photic zone, suggesting urea is widely available to the deep-sea microbiome globally. Our results demonstrate that urea is a nitrogen source to abundant and diverse microorganisms in the dark ocean, as well as a significant contributor to deep-sea nitrification and therefore fuel for chemoautotrophy.

Urea is hypothesized to be an important source of nitrogen and chemical energy to microorganisms in the deep sea; however, direct evidence for urea use below the epipelagic ocean is lacking. Here, we explore urea utilization from 50 to 4000 meters depth in the northeastern Pacific Ocean using metagenomics, nitrification rates, and single-cell stable-isotope-uptake measurements with nanoscale secondary ion mass spectrometry (nanoSIMS). We find that the majority (>60%) of active cells across all samples assimilated urea-derived N, and that cell-specific nitrogen-incorporation rates from urea were higher than that from ammonium. Both urea concentrations and assimilation rates relative to ammonium generally increased below the euphotic zone. We detected ammonia- and urea-based nitrification at all depths at one of two sites analyzed, demonstrating their potential to support chemoautotrophy in the mesopelagic and bathypelagic regions. Using newly generated metagenomes we find that the ureC gene , encoding the catalytic subunit of urease, is found within 39% of deep-sea cells in this region, including the Nitrosophaerota (likely for nitrification) as well as thirteen other phyla such as Proteobacteria, Verrucomicrobia, Plantomycetota, Nitrospinota, and Chloroflexota (likely for assimilation). Analysis of public metagenomes revealed ureC within 10-46% of deep-sea cells around the world, with higher prevalance below the photic zone, suggesting urea is widely available to the deep-sea microbiome globally. Our results demonstrate that urea is a nitrogen source to abundant and diverse microorganisms in the dark ocean, as well as a significant contributor to deep-sea nitrification and therefore fuel for chemoautotrophy.

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 and the isotopic labeling of the central (α) and terminal (β) nitrogen (N) 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 labeling of the two nitrogen atoms in the N2O molecule. Implementing the rates of labeled N2O production in a time-dependent numerical model, we found that N2O production from NO3- dominated at most stations and depths, with rates as high as 1600 ± 200 pM N2O d-1. Hybrid N2O production, one of the mechanisms by which ammonia-oxidizing archaea produce N2O, had rates as high as 230 ± 80 pM N2O d-1 that peaked in both the near-surface and deep N2O concentration maxima. Based on the equal production of 45N2Oα and 45N2Oβ in the majority of our experiments, we infer that hybrid N2O production likely has a consistent site preference, despite drawing from two distinct substrate pools. We also found that the rates and yields of hybrid N2O production were enhanced at low dissolved oxygen concentrations ([O2]), with hybrid N2O yields as high as 20 % at depths where [O2] was below detection (880 nM) but nitrification was still active. Finally, we identified a few incubations with [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.

Light is considered a strong controlling factor of nitrification rates in the surface ocean. Previous work has shown that ammonia oxidation and nitrite oxidation may be inhibited by high light levels, yet active nitrification has been measured in the sunlit surface ocean. While it is known that photosynthetically active radiation (PAR) influences microbial nitrite production and consumption, the level of inhibition of nitrification is variable across datasets. Additionally, phytoplankton have light-dependent mechanisms for nitrite production and consumption that co-occur with nitrification around the depths of the primary nitrite maximum (PNM). In this work, we experimentally determined the direct influence of light level on net nitrite production, including all major nitrite cycling processes (ammonia oxidation, nitrite oxidation, nitrate reduction and nitrite uptake) in microbial communities collected from the base of the euphotic zone. We found that although ammonia oxidation was inhibited at the depth of the PNM and was further inhibited by increasing light at all stations, it remained the dominant nitrite production process at most stations and treatments, even up to 25 % surface PAR. Nitrate addition did not enhance ammonia oxidation in our experiments but may have increased nitrate and nitrite uptake at a coastal station. In contrast to ammonia oxidation, nitrite oxidation was not clearly inhibited by light and sometimes even increased at higher light levels. Thus, accumulation of nitrite at the PNM may be modulated by changes in light, but light perturbations did not exclude nitrification from the surface ocean. Nitrite uptake and nitrate reduction were both enhanced in high-light treatments relative to low light and in some cases showed high rates in the dark. Overall, net nitrite production rates of PNM communities were highest in the dark treatments.

Because nitrogen availability limits primary production over much of the global ocean, understanding the controls on the marine nitrogen inventory and supply to the surface ocean is essential for understanding biological productivity and exchange of greenhouse gases with the atmosphere. Quantifying the ocean’s inputs, outputs, and internal cycling of nitrogen requires a variety of tools and approaches, including measurements of the nitrogen isotope ratio in organic and inorganic nitrogen species. The marine nitrogen cycle, which shapes nitrogen availability and speciation in the ocean, is linked to the elemental cycles of carbon, phosphorus, and trace elements. For example, the majority of nitrogen cycle oxidation and reduction reactions are mediated by enzymes that require trace metals for catalysis. Recent observations made through global-scale programs such as GEOTRACES have greatly expanded our knowledge of the marine nitrogen cycle. Though much work remains to be done, here we outline key advances in understanding the marine nitrogen cycle that have been achieved through these analyses, such as the distributions and rates of dinitrogen fixation, terrestrial nitrogen inputs, and nitrogen loss processes.

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.

The El Niño‐Southern Oscillation (ENSO) is a natural climate phenomenon that alters the biogeochemical and physical dynamics of the Eastern Tropical Pacific Ocean. Its two phases, El Niño and La Niña, are characterized by decreased and increased coastal upwelling, respectively, which have cascading effects on primary productivity, organic matter supply, and ocean‐atmosphere interactions. The Eastern Tropical South Pacific oxygen minimum zone is a source of nitrous oxide (N2O), a potent greenhouse gas, to the atmosphere. Here, we present the first study to directly compare N2O sources during opposing ENSO phases using N2O isotopocule analyses. Our data show that during La Niña, N2O accumulation increased six‐fold in the upper 100 m of the water column, and N2O fluxes to the atmosphere increased up to 20‐fold. N2O isotopocule data demonstrated substantial increases in δ¹⁸O up to 60.5‰ and decreases in δ¹⁵Nβ down to −10.3‰ in the oxycline, signaling a shift in N2O cycling during La Niña compared to El Niño. During El Niño, N2O production was primarily due to ammonia‐oxidizing archaea, whereas during La Niña, N2O production by incomplete denitrification supplemented that from ammonia‐oxidation, with N2O consumption likely maintaining the high site preference values (up to 26.7‰). Ultimately, our results illustrate a strong connection between upwelling intensity, biogeochemistry, and N2O flux to the atmosphere. Additionally, they highlight the combined power of N2O isotopocule analysis and repeat measurements in the same region to constrain N2O interannual variability and cycling dynamics under different climate scenarios.