Andrew R. Babbin

Organic Matter Stoichiometry, Flux, and Oxygen Control Nitrogen Loss in the Ocean

Understanding N Loss Biologically available nitrogen (N) is essential for marine plants, and shortage of N limits photosynthesis. Marine N can be removed by denitrification and anaerobic ammonia oxidation (anammox) processes, but what controls the balance between these two pathways? Babbin et al. (p. 406, published online 10 April) tested the effects of stoichiometry on N removal in the lab and found that the balance of N loss processes depends on the stoichiometry of the source organic material. The variable ratio of denitrification to anammox in the ocean is due to variations in organic matter quality and quantity. Biologically available nitrogen limits photosynthesis in much of the world ocean. Organic matter (OM) stoichiometry had been thought to control the balance between the two major nitrogen removal pathways—denitrification and anammox—but the expected proportion of 30% anammox derived from mean oceanic OM is rarely observed in the environment. With incubations designed to directly test the effects of stoichiometry, however, we showed that the ratio of anammox to denitrification depends on the stoichiometry of OM supply, as predicted. Furthermore, observed rates of nitrogen loss increase with the magnitude of OM supply. The variable ratios between denitrification and anammox previously observed in the ocean are thus attributable to localized variations in OM quality and quantity and do not necessitate a revision to the global nitrogen cycle.

Rapid nitrous oxide cycling in the suboxic ocean

More N2O is no laughing matter Because N2O is a potent greenhouse gas, tracking its sources and sinks—including those from natural processes—is imperative. Babbin et al. developed an isotopic tracer method to measure biological N2O reduction rates directly in the Eastern Tropical North Pacific Ocean. Incomplete denitrification results in the rapid cycling and net accumulation of N2O. As oxygen minimum zones expand in the global ocean, more N2O may enter the atmosphere than previously expected. Science, this issue p. 1127 Nitrous oxide undergoes rapid biological cycling in the ocean’s oxygen minimum zones. Nitrous oxide (N2O) is a powerful greenhouse gas and a major cause of stratospheric ozone depletion, yet its sources and sinks remain poorly quantified in the oceans. We used isotope tracers to directly measure N2O reduction rates in the eastern tropical North Pacific. Because of incomplete denitrification, N2O cycling rates are an order of magnitude higher than predicted by current models in suboxic regions, and the spatial distribution suggests strong dependence on both organic carbon and dissolved oxygen concentrations. Furthermore, N2O turnover is 20 times higher than the net atmospheric efflux. The rapid rate of this cycling coupled to an expected expansion of suboxic ocean waters implies future increases in N2O emissions.

Enhancement of anammox by the excretion of diel vertical migrators

Significance Nitrogen, the limiting nutrient for primary production across much of the ocean, is converted to biologically inactive N2 by denitrification and anaerobic ammonium oxidation (anammox) in anoxic waters. Anammox requires an active source of ammonium, which can be provided by concurrent denitrification. However, anammox has been observed at high rates in the absence of denitrification, and the source of ammonium has remained cryptic. Using a combination of observations and models, we suggest that zooplankton and micronekton provide a missing source of ammonium to anoxic waters through diel vertical migrations, fueling anammox and decoupling it from denitrification. This previously overlooked mechanism can help to reconcile observations with theory and highlights the role of animals on ocean biogeochemistry. Measurements show that anaerobic ammonium oxidation with nitrite (anammox) is a major pathway of fixed nitrogen removal in the anoxic zones of the open ocean. Anammox requires a source of ammonium, which under anoxic conditions could be supplied by the breakdown of sinking organic matter via heterotrophic denitrification. However, at many locations where anammox is measured, denitrification rates are small or undetectable. Alternative sources of ammonium have been proposed to explain this paradox, for example through dissimilatory reduction of nitrate to ammonium and transport from anoxic sediments. However, the relevance of these sources in open-ocean anoxic zones is debated. Here, we bring to attention an additional source of ammonium, namely, the daytime excretion by zooplankton and micronekton migrating from the surface to anoxic waters. We use a synthesis of acoustic data to show that, where anoxic waters occur within the water column, most migrators spend the daytime within them. Although migrators export only a small fraction of primary production from the surface, they focus excretion within a confined depth range of anoxic water where particle input is small. Using a simple biogeochemical model, we suggest that, at those depths, the source of ammonium from organisms undergoing diel vertical migrations could exceed the release from particle remineralization, enhancing in situ anammox rates. The contribution of this previously overlooked process, and the numerous uncertainties surrounding it, call for further efforts to evaluate the role of animals in oxygen minimum zone biogeochemistry.

Nitrous oxide production by nitrification and denitrification in the Eastern Tropical South Pacific oxygen minimum zone

The Eastern Tropical South Pacific oxygen minimum zone (ETSP-OMZ) is a site of intense nitrous oxide (N2O) flux to the atmosphere. This flux results from production of N2O by nitrification and denitrification, but the contribution of the two processes is unknown. The rates of these pathways and their distributions were measured directly using 15N tracers. The highest N2O production rates occurred at the depth of peak N2O concentrations at the oxic-anoxic interface above the oxygen deficient zone (ODZ) because slightly oxygenated waters allowed (1) N2O production from both nitrification and denitrification and (2) higher nitrous oxide production yields from nitrification. Within the ODZ proper (i.e., anoxia), the only source of N2O was denitrification (i.e., nitrite and nitrate reduction), the rates of which were reflected in the abundance of nirS genes (encoding nitrite reductase). Overall, denitrification was the dominant pathway contributing the N2O production in the ETSP-OMZ.

Connecting the dots: linking nitrogen cycle gene expression to nitrogen fluxes in marine sediment mesocosms

Connecting molecular information directly to microbial transformation rates remains a challenge, despite the availability of molecular methods to investigate microbial biogeochemistry. By combining information on gene abundance and expression for key genes with quantitative modeling of nitrogen fluxes, we can begin to understand the scales on which genetic signals vary and how they relate to key functions. We used quantitative PCR of DNA and cDNA, along with biogeochemical modeling to assess how the abundance and expression of microbes responsible for two steps in the nitrogen cycle changed over time in estuarine sediment mesocosms. Sediments and water were collected from coastal Massachusetts and maintained in replicated 20 L mesocosms for 45 days. Concentrations of all major inorganic nitrogen species were measured daily and used to derive rates of nitrification and denitrification from a Monte Carlo-based non-negative least-squares analysis of finite difference equations. The mesocosms followed a classic regeneration sequence in which ammonium released from the decomposition of organic matter was subsequently oxidized to nitrite and then further to nitrate, some portion of which was ultimately denitrified. Normalized abundances of ammonia oxidizing archaeal ammonia monoxoygenase (amoA) transcripts closely tracked rates of ammonia oxidation throughout the experiment. No such relationship, however, was evident between denitrification rates and the normalized abundance of nitrite reductase (nirS and nirK) transcripts. These findings underscore the complexity of directly linking the structure of the microbial community to rates of biogeochemical processes.

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.

Vertical modeling of the nitrogen cycle in the eastern tropical South Pacific oxygen deficient zone using high-resolution concentration and isotope measurements

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.

Nitrogen substrate–dependent nitrous oxide cycling in salt marsh sediments

Nitrous oxide (N2O) is important to Earth’s climate because it is a strong absorber of radiation and an important ozone depletion agent. Increasing anthropogenic nitrogen input into the marine environment, especially to coastal waters, has led to increasing N2O emissions. Identifying the nitrogen compounds that serve as substrates for N2O production in coastal waters reveals important pathways and helps us understand their control by environmental factors. In this study, sediments were collected from a long-term fertilization site in Great Sippewissett Marsh, Falmouth, Massachusetts. The 15N tracer incubation time course experiments were conducted and analyzed for potential N2O production and consumption rates. The two nitrogen substrates of N2O production, ammonium and nitrate, correspond to the two production pathways, nitrification and denitrification, respectively. When measurable nitrate was present, despite ambient high ammonium concentrations, denitrification was the major N2O production pathway. When nitrate was absent, ammonium became the dominant substrate for N2O production, via nitrification and coupled nitrification-denitrification. Net N2O consumption was enhanced under low oxygen and nitrate conditions. N2O production and consumption rates increased with increasing levels of nitrogen fertilization in long-term experimental plots. These results indicate that increasing anthropogenic nitrogen input to salt marshes can stimulate sedimentary N2O production via both nitrification and denitrification, whereas episodic oxygen depletion results in net N2O consumption.

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 15N 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.