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Gayana (Concepción)

versión impresa ISSN 0717-652Xversión On-line ISSN 0717-6538

Gayana (Concepc.) v.70  supl.1 Concepción oct. 2006 

Suplemento Gayana 70: 14-18, 2006


Nitrogen cycling in sub-oxic water colmns


Ciclamiento del nitrógeno en columnas de agua sub-oxicas


Tage Dalsgaard1, Bo Thamdrup2 & Marlene Mark Jensen2


1. National Environmental Research Institute, Department of Marine Ecology, Silkeborg, Denmark,

2. Danish Center for Earth System Science, Institute of Biology, University of Southern Denmark, Odense M, Denmark


The current knowledge about nitrogen removal processes in suboxic water columns will be reviewed. The most recent development in the understanding of these processes is the documentation of anaerobic ammonium oxidation with nitrite (anammox) in these areas, and the balance between the traditional denitrification and anammox will be in focus.

Keywords: Denitrification, anammox, water column, nitrogen removal.


Se revisa el conocimiento actual sobre los procesos de remoción del nitrógeno en columnas de agua subóxicas. En estas áreas el más reciente desarrollo en la comprensión de estos procesos es la documentación sobre la oxidación anaeróbica del amonio con nitrato (anammox). Además enfocaremos el balance entre la tradicional denitrificación y el anammox .

Palabras Claves: Denitrificación, anammox, columna de agua, remoción del nitrógeno.


In the three main oxygen minimum zones (OMZ) of the world, the Eastern Tropical North and South Pacific and the Arabian Sea, an intense nitrogen removal is believed to occur. By current estimates these OMZ's are responsible for about 1/3 of the global marine nitrogen removal (Codispoti et al. 2001) despite the fact that they cover less than 2% of the ocean surface area. Marine sediments are responsible for the remaining 2/3 of the nitrogen removal. In the literature marine nitrogen removal has been estimated in several often indirect ways and has been assigned `denitrification' (e.g., Brandes & Devol 2002, Codispoti et al. 2001, Codispoti et al. 1986, Deutsch et al. 2001). However, early on it was suggested that maybe not all nitrogen removal in the water column of the OMZ's was due to denitrification. More nitrogen was missing from anoxic water columns than could be explained by traditional denitrification (Richards 1965a, Richards 1965b, Richards et al. 1965). Assuming that denitrification was the main mineralization process in the OMZ, and that the organic matter undergoing mineralization contained C, N and P in the Redfield ratio (Redfield et al. 1960), ammonium should be produced at a rate 16 times faster than phosphate according to Eq. 1:

(CH2O)106(NH 3)16H3PO4 + 84.8HNO3 g

106CO2 + 16NH3 + 42.4N2 + 148.4H2O + H3PO4 1)

However, it was repeatedly shown that ammonium was almost undetectable in the OMZ, and since ammonium could not be converted via nitrification in the absence of oxygen, Richards suggested that ammonium was oxidised anaerobically with nitrate to N2. If this was occurring together with denitrification it would explain the absence of ammonium accumulation according to the following reaction:

(CH2O)106(NH 3)16H3PO4 + 94.4HNO3 g 106CO2 + 55.2N2 + 177.2H2O + H3PO4 2)

The anaerobic oxidation of ammonium with nitrate (or nitrite) suggested above had not been demonstrated to occur and it was not until 1995 that this process was documented (Mulder et al. 1995). It was found in a waste water treatment plant and named "anammox". It soon became clear that nitrite was the direct oxidant of ammonium (Van De Graaf et al. 1995) and it occurred as a 1:1 reaction between nitrite and ammonium according to Eq. 3:

NO2- + NH4+ g N2 + 2H2O 3)

The process was performed by a specialized group of bacteria, the Planctomycetes (Strous et al. 1999). Hydroxylamine and hydrazine appear to be intermediates of the process which occur in a membrane bound compartment, the anammoxosome. The membrane consists of a unique type of lipids, the ladderane lipids, which may be used to identify the presence of anammox bacteria in the environment.

A few years later the anammox process was discovered in nature and found to be important for nitrogen removal in continental shelf sediments (Thamdrup & Dalsgaard 2002) and in some sediments it even exceeded the traditional denitrification. The relative importance of anammox for nitrogen gas production apparently increases with water depth, at least until a depth of 700 m, which is the deepest site sampled so far (Dalsgaard et al. 2005). Soon after the discovery in sediments it was also found in suboxic water columns: in the coastal bay Golfo Dulce in Costa Rica (Dalsgaard et al. 2003), in the anoxic waters of the Black Sea (Kuypers et al. 2003) and in the suboxic water column in the Benguela upwelling system off Namibia (Kuypers et al. 2005) and latest also in the OMZ off Iquique in Chile (Thamdrup et al. in press). In this presentation the evidence for the occurrence of anammox and denitrification in the above mentioned papers will be reviewed.


Golfo Dulce

The water chemistry of Golfo Dulce is a good example of a water column where NH4+ is missing. According to the traditional understanding of mineralization in such a water column there should be 16 times as much NH4+ as PO43- (Eq. 1). That was clearly not the case here and NH4+ remained low down through the water column despite the fact that PO43- increased (Fig. 1). The missing NH4+ alone is an indication of anaerobic ammonium oxidation.

Figure 1. Water chemistry at st. B in Golfo Dulce, Costa Rica. After Dalsgaard et al., 2003.


Through 15N-labelling experiments it was demonstrated that it was an anammox-like reaction that was responsible for the removal of NH4+ (Dalsgaard et al. 2003). In experiments with 15NH4+ added 14N15N was formed immediately (Fig. 2a) indicating oxidation of the 15N in NH4+ to N2. This is an indication of the anammox reaction between 15N from NH4+ and 14N from the native unlabelled NO3-/NO2- pool. When 15NO3- was added both 14N15N and 15N15N were produced after an initial lag phase (Fig. 2b). Knowing the 15N

Figure 2. Examples of the production of 15N-labelled N2 from different treatments as indicated. After Dalsgaard et al., 2003.

content in the NO3- pool the expected 14N15N production by denitrification could be estimated (Thamdrup & Dalsgaard 2002). However, the actual measured 14N15N production was considerably higher than that from denitrification, indicating another source of 14N15N consistent with the anammox reaction. In parallel experiment where both 15NO3- and 14NH4+ were added, 14N15N production was even higher, indicating NH4+ limitation of the anammox. The stimulation of anammox by NH4+ addition was a factor of 4 and 2 for Station A and B respectively. This would indicate that anammox is able to keep the NH4+ concentration low and that the anammox reaction is a good explanation for the missing NH4+ as suggested already in 1965 (see above).

Both anammox and denitrification were detected at all four sampling depths at each of the two stations in Golfo Dulce (Fig. 3). The most consistent variation in process rates was that denitrification increased towards the bottom, probably as a result of increased availability of reduced species, such as sulfide, that acted as electron donor for denitrification. The relative importance of anammox for N2-production also varied quite significantly from 13-68%. The average for the upper three sampling depths, which were not influenced by the sediment and thus resemble pelagic oxygen minimum zones, were 58% and 32% for station A and B, respectively. Eq. 2 predicts that 16 of the 55.2 N2 molecules (29%) are produced by anammox, if the two processes are as intimately coupled as indicated by the equation. The fact that a higher proportion of the N2 was produced by anammox may be explained by a preferential degradation of N-rich organic matter, which would increase the NH4+ production by denitrification.

The Black Sea

Almost simultaneously with the finding of anammox in the water column of Golfo Dulce the process was found in the anoxic water column of the Black Sea (Kuypers et al. 2003). The anammox process was documented by 15N tracer experiments and anammox bacteria were shown to be present using molecular techniques and through identification of ladderane lipids. Model calculations showed that the anammox process potentially could be responsible for oxidation of all the NH4+ transported up towards the NO3- containing waters.

Figure 3. Rates of anammox and denitrification in Golfo Dulce, Costa Rica (bars) and the relative contribution of anammox to N2 production. After Dalsgaard et al., 2003.

The Benguela upwelling system

Kuypers and co-workers (2005) investigated the mechanisms of nitrogen removal in the Benguela upwelling system off Namibia. They found massive losses of fixed nitrogen in the suboxic waters over the continental shelf. They were unable to demonstrate denitrification but found instead that anammox was responsible for all of the nitrogen removal. A very interesting finding, considering that denitrification, until recently, has been regarded as the sole process removing fixed nitrogen from the ocean. The anammox rates increased towards the sediment, and correlated to some degree with the turbidity of the water, indicating that the anammox process may partially be linked to resuspended sediments. As in the Black Sea study the presence of anammox bacteria was demonstrated clearly with molecular techniques and through identification of the ladderane lipids.

Eastern Tropical South Pacific

A similar result was found in the OMZ off Iquique in the northern Chile. Here Thamdrup and co-workers (in press) also found anammox to be the dominating N2 producing process and denitrification was only found in one out of six incubations where anammox was found. During the first 24 hours of the incubation there was linear increase in the anammox produced N2, and, in one instance, also in denitrification produced N2. After 36 - 48 hours rates generally increased and denitrification was detectable in most incubations. During this last part of the incubations a rapid reduction of NO3- to NO2- was also observed. The processes and rates estimated from the first 24 hours of incubation are interpreted as in situ rates and the acceleration of rates later is explained as artifacts developing during the incubation. However, it means that the potential for both nitrate reduction and denitrification was present in the water column. However, this can not be a persistent situation as this would require unrealistic C:N ratios in the organic matter undergoing mineralization. It is also suggested that the mineralization occurring in the water at the time of sampling was reduction of NO3- to NO2-. This would mean that denitrifiers, and other bacteria capable of reducing NO3-, mineralize the organic matter without N2-gas production. The NH4+ and NO2- produced by the mineralization would then be available for N2 production through anammox.


The recent measurements cited above indicate that denitrification can no longer be viewed as synonymous with nitrogen removal in suboxic water columns, and that anammox plays a significant role in the N2 production. In the two studies from the open ocean it even appears that anammox may be responsible for all of the N2 production. It is also clear, from stoichiometric considerations, that, overall, anammox can not be responsible for all of the N2 production. Temporal separation of the different steps in the denitrification process may help explain these results. It is thus proposed that mineralization of organic matter in the suboxic water column may be seen as a succession of processes where the relative importance of anammox and denitrification may vary over time.


This work was supported by the Danish National Science Council and the Swedish Foundation for Strategic Environmental Research (MISTRA). We thank Osvaldo Olloa and his team for arranging the Cruise off Iquique in Chile and Jenaro Acuña-Gonzáles and José A. Vargas for the collaboration for the work in Golfo Dulce in Costa Rica.


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