Tage Dalsgaard

Production of N2 through Anaerobic Ammonium Oxidation Coupled to Nitrate Reduction in Marine Sediments

ABSTRACT In the global nitrogen cycle, bacterial denitrification is recognized as the only quantitatively important process that converts fixed nitrogen to atmospheric nitrogen gas, N2, thereby influencing many aspects of ecosystem function and global biogeochemistry. However, we have found that a process novel to the marine nitrogen cycle, anaerobic oxidation of ammonium coupled to nitrate reduction, contributes substantially to N2 production in marine sediments. Incubations with 15N-labeled nitrate or ammonium demonstrated that during this process, N2 is formed through one-to-one pairing of nitrogen from nitrate and ammonium, which clearly separates the process from denitrification. Nitrite, which accumulated transiently, was likely the oxidant for ammonium, and the process is thus similar to the anammox process known from wastewater bioreactors. Anaerobic ammonium oxidation accounted for 24 and 67% of the total N2 production at two typical continental shelf sites, whereas it was detectable but insignificant relative to denitrification in a eutrophic coastal bay. However, rates of anaerobic ammonium oxidation were higher in the coastal sediment than at the deepest site and the variability in the relative contribution to N2 production between sites was related to large differences in rates of denitrification. Thus, the relative importance of anaerobic ammonium oxidation and denitrification in N2 production appears to be regulated by the availability of their reduced substrates. By shunting nitrogen directly from ammonium to N2, anaerobic ammonium oxidation promotes the removal of fixed nitrogen in the oceans. The process can explain ammonium deficiencies in anoxic waters and sediments, and it may contribute significantly to oceanic nitrogen budgets.

N2 production by the anammox reaction in the anoxic water column of Golfo Dulce, Costa Rica.

In oxygen-depleted zones of the open ocean, and in anoxic basins and fjords, denitrification (the bacterial reduction of nitrate to give N2) is recognized as the only significant process converting fixed nitrogen to gaseous N2. Primary production in the oceans is often limited by the availability of fixed nitrogen such as ammonium or nitrate, and nitrogen-removal processes consequently affect both ecosystem function and global biogeochemical cycles. It was recently discovered that the anaerobic oxidation of ammonium with nitrite—the ‘anammox’ reaction, performed by bacteria—was responsible for a significant fraction of N2 production in some marine sediments. Here we show that this reaction is also important in the anoxic waters of Golfo Dulce, a 200-m-deep coastal bay in Costa Rica, where it accounts for 19–35% of the total N2 formation in the water column. The water-column chemistry in Golfo Dulce is very similar to that in oxygen-depleted zones of the oceans—in which one-half to one-third of the global nitrogen removal is believed to occur. We therefore expect the anammox reaction to be a globally significant sink for oceanic nitrogen.

A Cryptic Sulfur Cycle in Oxygen-Minimum–Zone Waters off the Chilean Coast

Cryptic Sulfur Cycling Aerobic bacteria and ocean circulation patterns control the formation and distribution of oxygen-minimum zones at moderate depth in the oceans. These habitats host microorganisms that thrive on other metabolic substrates in the absence of oxygen—most commonly, metabolizing thermodynamically favorable nitrogen compounds like nitrate. Off the coast of Chile, however, Canfield et al. (p. 1375, published online 11 November; see the Perspective by Teske) suggest that bacteria may often reduce sulfate as well. Metagenomic sequencing revealed the presence of both sulfate-reducing and sulfide-oxidizing bacteria. With the coincidence of sulfate and nitrate reduction, the sulfur and nitrogen cycles may be intimately linked; for example, sulfate reduction could provide nitrogen-rich ammonium for bacteria that ultimately transform it into nitrogen gas. Bacterial sulfur reduction and oxidation accompanies nitrogen cycling where oxygen levels at depth are low. Nitrogen cycling is normally thought to dominate the biogeochemistry and microbial ecology of oxygen-minimum zones in marine environments. Through a combination of molecular techniques and process rate measurements, we showed that both sulfate reduction and sulfide oxidation contribute to energy flux and elemental cycling in oxygen-free waters off the coast of northern Chile. These processes may have been overlooked because in nature, the sulfide produced by sulfate reduction immediately oxidizes back to sulfate. This cryptic sulfur cycle is linked to anammox and other nitrogen cycling processes, suggesting that it may influence biogeochemical cycling in the global ocean.

Factors Controlling Anaerobic Ammonium Oxidation with Nitrite in Marine Sediments

ABSTRACT Factors controlling the anaerobic oxidation of ammonium with nitrate and nitrite were explored in a marine sediment from the Skagerrak in the Baltic-North Sea transition. In anoxic incubations with the addition of nitrite, approximately 65% of the nitrogen gas formation was due to anaerobic ammonium oxidation with nitrite, with the remainder being produced by denitrification. Anaerobic ammonium oxidation with nitrite exhibited a biological temperature response, with a rate optimum at 15°C and a maximum temperature of 37°C. The biological nature of the process and a 1:1 stoichiometry for the reaction between nitrite and ammonium indicated that the transformations might be attributed to the anammox process. Attempts to find other anaerobic ammonium-oxidizing processes in this sediment failed. The apparent Km of nitrite consumption was less than 3 μM, and the relative importance of ammonium oxidation with nitrite and denitrification for the production of nitrogen gas was independent of nitrite concentration. Thus, the quantitative importance of ammonium oxidation with nitrite in the jar incubations at elevated nitrite concentrations probably represents the in situ situation. With the addition of nitrate, the production of nitrite from nitrate was four times faster than its consumption and therefore did not limit the rate of ammonium oxidation. Accordingly, the rate of this process was the same whether nitrate or nitrite was added as electron acceptor. The addition of organic matter did not stimulate denitrification, possibly because it was outcompeted by manganese reduction or because transport limitation was removed due to homogenization of the sediment.

Effects of salinity on NH4+ adsorption capacity, nitrification, and denitrification in Danish estuarine sediments

The regulatory effect of salinity on nitrogen dynamics in estuarine sediments was investigated in the Randers Fjord estuary, Denmark, using sediment slurries and intact sediment cores and applying 15N-isotope techniques. Sediment was sampled at three representative stations varying in salinity, and all experiments were run at 0‰, 10‰, 20‰, and 30‰. The sediment NH4+ adsorption capacity decreased markedly at all stations when salinity was increased from 0‰ to 10‰; further increase showed little effect. In situ nitrification and denitrification also decreased with increasing salinities, with the most pronounced reduction of approximately 50% being observed when the salinity was raised from 0‰ to 10‰. The salinity-induced reduction in NH4+ adsorption capacity and stimulation of NH4+ efflux has previously been argued to cause a reduction in nitrification activity since the nitrifying bacteria become limited by NH4+ availability at higher salinities. However, using a potential nitrification assay where NH4+ was added in excess, it was demonstrated that potential nitrification activity also decreased with increasing salinity, indicating that the inhibitory salinity effect may also be a physiological effect on the microorganisms. This hypothesis was supported by the finding that denitrification based on NO3− from the overlying water (Dw), which is independent of the nitrification process, and hence NH4+ availability, also decreased with increasing salinity. We conclude that changes in salinity have a significant effect on nitrogen dynamics in estuarine sediments, which must be considered when nitrogen transformations are measured and evaluated.

LONG‐TERM CHANGES AND IMPACTS OF HYPOXIA IN DANISH COASTAL WATERS

A 38-year record of bottom water dissolved oxygen concentrations in coastal marine ecosystems around Denmark (1965-2003) and a longer partially reconstructed record of total nitrogen (TN) inputs (1900-2003) were assembled to describe long-term patterns in hypoxia and anoxia. Interannual variations in bottom water oxygen concentrations were analyzed in relation to various explanatory variables (bottom temperature, wind speed, advective transport, TN loading). Reconstructed TN loads peaked in the 1980s with a gradual decline to the present, commensurate with a legislated nutrient reduction strategy. Mean bottom water oxygen concentrations during summer have significantly declined in coastal marine ecosystems, decreasing substantially during the 1980s and were extremely variable thereafter. Despite decreasing TN loads, the worst hypoxic event ever recorded in open waters occurred in 2002. For estuaries and coastal areas, bottom water oxygen concentrations were best described by TN input from land and wind speed in July-September, explaining 52% of the interannual variation in concentrations. For open sea areas, bottom water oxygen concentrations were also modulated by TN input from land, however, additional significant variables included advective transport of water and Skagerrak surface water temperature and explained 49% of interannual variations in concentrations. Reductions in benthic species number and alpha diversity were significantly related to the duration of the 2002 hypoxic event. Gradual decreases in diversity measures (species number and alpha diversity) over the first 2-4 weeks show that the benthic community undergoes significant changes before the duration of hypoxia is severe enough to cause the community to collapse. Enhanced sediment-water fluxes of NH4+ and PO43- occur with hypoxia, increasing nutrient concentrations in the water column, and stimulating additional phytoplankton production. Repeated hypoxic events have changed the character of benthic communities and how organic matter is processed in sediments. Our data suggest that repeated hypoxic events lead to an increase in susceptibility of Danish waters to eutrophication and further hypoxia. (Less)

Comparison of isotope pairing and N 2 /Ar methods for measuring sediment dentrification - assumptions, modifications and implications

Denitrification has been measured during the last few years using two different methods in particular: isotope pairing measured on a triple-collector isotopic ratio mass spectrometer and N2:Ar ratios measured on a membrane inlet mass spectrometer (MIMS). This study compares these two techniques in short-term batch experiments. Rates obtained using the original N2∶Ar method were up to 3 to 4 times higher than rates obtained using the isotope pairing technique due to O2 reacting with the N2 during MIMS analysis. Oxygen combines with N2 within the mass spectrometer ion source forming NO+ which reduces the N2 concentration. The decrease in N2 is least at lower O2 concentrations and since oxygen is typically consumed during incubations of sediment cores, the result is often a pseudo-increase in N2 concentration being interpreted as denitrification activity. The magnitude of this ocygen effect may be instrument specific. The reaction of O2 with N2 and the subsequent decrease in N2 was only partly correctly using an O2 correction curve for the relationship between N2 and O2 concentrations. The O2 corrected N2∶Ar denitrification rates were lower, but still did not match the isotope pairing rates and the variability between replicates was much higher. Using a copper reduction column heated to 600°C to remove all of the O2 from the sample before MIMS analysis resulted in comparable rates (slightly lower), and comparable variability between replicates, to the isotope pairing technique. The N2:Ar technique determines the net N2 production as the difference between N2 production by denitrification and N2 consumption by N-fixation, while N-fixation has little effect on the isotope pairing technique which determines a rate very close to the gross N2 production. When the two different techniques were applied on the same sediment, the small difference in rates obtained by the two methods seemed to reflect N-fixation as also supported from measurements of ethylene production in acetylene enriched sediment cores. The N2:Ar and isotope pairing techniques may be combined to provide simultaneous measurements of denitrification and N-fixation. Both techniques have several assumptions that must be met to achieve accurate rates; a number of tests are outlined that can be applied to demonstrate that these assumptions are being meet.

Impacts of longline mussel farming on oxygen and nitrogen dynamics and biological communities of coastal sediments

Benthic communities and benthic mineralization were studied in two shallow coastal regions of New Zealand: Tasman Bay, a possible future site for mussel farm development, and Beatrix Bay, which already hosts several longline mussel farms. In Tasman Bay, microphytobenthic (MPB) production added significantly to the total primary production of the bay. The activity of benthic microalgae had a pronounced effect on oxic conditions, solute exchange and denitrification rates. Benthic mineralization, quantified as the dark oxygen uptake, was in the range of 675±11 μmol m−2 h−1. Denitrification rates were high and fueled entirely by nitrate produced by the nitrifying community within the sediment. Competition for inorganic nitrogen between benthic microalgae and nitrifiers/denitrifiers resulted in diel variation in nitrogen cycling and reduced the inorganic nitrogen efflux and denitrification activity in the light. Calculated in electron equivalents, denitrification accounted for 11–20% of the total carbon mineralization—one of the highest numbers reported for coastal sediments. Reduced sediments, containing low MPB biomass and few subsurface macroinvertebrate species, were observed below a mussel farm in Beatrix Bay, presumably due to the intensified sedimentation of organic matter. Oxygen consumption increased in the organic-rich sediments, and ammonium effluxes were up to 14 times higher than those of unaffected sediments 250 m away from the farm. Denitrification rates below the farm were low as the coupled nitrification–denitrification was inhibited by the presence of sulfide. The dissimilative reduction of nitrate to ammonium (DRNA) was, however, stimulated in the reduced sediment. The enhanced benthic mineralization was associated with sulfidic sediments and a lower nitrogen removal rate due to impeded benthic photosynthesis and denitrification activity. The described local conditions associated with mussel farming should be taken into account when new areas are considered for development.

Effects of fish farm waste on Posidonia oceanica meadows: Synthesis and provision of monitoring and management tools

This paper provides a synthesis of the EU project MedVeg addressing the fate of nutrients released from fish farming in the Mediterranean with particular focus on the endemic seagrass Posidonia oceanica habitat. The objectives were to identify the main drivers of seagrass decline linked to fish farming and to provide sensitive indicators of environmental change, which can be used for monitoring purposes. The sedimentation of waste particles in the farm vicinities emerges as the main driver of benthic deterioration, such as accumulation of organic matter, sediment anoxia as well as seagrass decline. The effects of fish farming on P. oceanica meadows are diverse and complex and detected through various metrics and indicators. A safety distance of 400 m is suggested for management of P. oceanica near fish farms followed by establishment of permanent seagrass plots revisited annually for monitoring the health of the meadows.

The fate of ammonium in anoxic manganese oxide-rich marine sediment

The possibility for anaerobic NH4+ oxidation and N2 formation was explored in a Mn oxide-rich continental basin sediment from Skagerrak. The surface sediment contained 2.9 weight-% Mn(IV), and reactive Mn oxide persisted to ≥10 cm depth. Microbial Mn reduction completely dominated anaerobic carbon oxidation, whereas neither Fe reduction nor sulfate reduction were significant. Accumulation rates of soluble NH4+ during anoxic incubations scaled with Mn reduction rates and did not indicate any substantial oxidation of NH4+. No sustained production of 15N-labelled N2 from added 15NH4+ was detectable during the four-day incubations, which constrains the rate of NH4+ conversion to N2 to <2% of the NH4+ production rate. Traces of 15N-labelled N2 accumulated initially, and this transient N2 production was possibly related to brief coupled nitrification/denitrification resulting from sediment handling. Oxidation of NH4+ to NO3− was also insignificant as there was no accumulation of NO3− during the incubations and added 15NO3− was rapidly consumed with N2 as a major product. Although the oxidation of NH4+ with Mn oxide is thermodynamically favorable, our results demonstrate that such oxidation was insignificant and that NH4+ can be considered the end product of nitrogen mineralization in this anoxic Mn oxide-rich sediment.