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2005 Anaerobic ammonium oxidation (anammox) in the marine environment


Research in Microbiology 156 (2005) 457–464 www.elsevier.com/locate/resmic

Mini-review

Anaerobic ammonium oxidation (anammox) in the marine environment
Tage Dalsgaard a,? , Bo Thamdrup b , Donald E. Can?eld b
a National Environmental Research Institute, Department of Marine Ecology, Vejls?vej 25, P.O. Box 314, 8600 Silkeborg, Denmark b Danish Centre for Earth System Science, Institute of Biology, University of Southern Denmark, Campusvej 55, 5230 Odense M, Denmark

Received 27 October 2004; accepted 19 January 2005 Available online 17 March 2005

Abstract Anammox, anaerobic ammonium oxidation with nitrite, is now recognized as an important process in the marine nitrogen cycle. The bacteria conducting anammox are highly specialized and appear to belong to the Planctomycetales. The process has now been found in a range of environments including marine sediments, sea ice and anoxic water columns, and it may be responsible for up to 50% of the global removal of ?xed nitrogen from the oceans. ? 2005 Elsevier SAS. All rights reserved.
Keywords: Anaerobic ammonium oxidation; Anammox; Review

1. Introduction Already decades ago, there were several indications that NH+ 4 could be oxidized anaerobically in nature. For example, in 1941 anaerobic NH+ 4 oxidation (now called anammox reduction as explained in Section 3) when coupled to NO? 2 was advanced as a possible source of N2 in the sea [11]. Later observations from anoxic water columns indicated that NH+ 4 disappeared under anoxic conditions [3,20,21], and the ? oxidation of NH+ 4 with NO3 to form N2 was the explanation most consistent with the data. Similar observations were made in the anoxic sul?de-free portion of the water column in the Black Sea [18]. Here, NH+ 4 transported from below disappeared before it met the O2 in the overlying water, and there was a ca. 20 m thick zone free of both O2 , NH+ 4 and H2 S. Nitrate was present here, and it was suggested that ? + NH+ 4 was oxidized with NO3 , preventing the NH4 from meeting the O2 . In marine sediments the oxidation of NH+ 4 with NO? had also been invoked to explain the anoxic dis3 appearance of NH+ 4 [1,16,27]. Moreover, calculations of G ? values for the oxidation of NH+ 4 with NO3 showed that the
* Corresponding author.

process was energetically favorable [2,3], yet another reason to expect it might occur in nature. In the early 1990’s the ?rst direct evidence of the anaerobic oxidation of NH+ 4 came from a waste water treatment facility in Delft, The Netherlands [36]. It proved dif?cult to isolate the process in the laboratory, but after a longer period of trial and error anammox was running in a laboratory-scale ?uidized bed reactor [17]. A few years after the initial discovery of anammox, the ?rst direct evidence for anaerobic NH+ 4 oxidation in a natural environment, marine sediments, was published [33]. Also, the initial suggestions of anaerobic NH+ 4 oxidation in anoxic water columns (e.g., [20]) have been corroborated by the discovery of anammox in the anoxic water column of a coastal bay in Costa Rica, Golfo Dulce [6], and in the anoxic water column of the Black Sea [15]. Here, we will review the current knowledge about the anammox process in the marine environment, also drawing from knowledge on anammox in waste water treatment. 2. The anammox bacteria 2.1. Cell biology Anammox bacteria are extremely dif?cult to isolate and thus far no pure cultures have been obtained. However,

E-mail address: tda@dmu.dk (T. Dalsgaard). 0923-2508/$ – see front matter ? 2005 Elsevier SAS. All rights reserved. doi:10.1016/j.resmic.2005.01.011

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some highly enriched cultures have been established from waste water treatment facilities, and from these we understand a great deal of both the biochemistry and the cell biology of the bacteria. All known anammox bacteria have a membrane-bound compartment in the cell, known as the anammoxosome, in which the anammox process is believed to take place [13,38]. The membrane of the anammoxosome contains ladderane lipids which form an exceptionally tight barrier against diffusion and which appear to be unique to anammox bacteria [8]. The hypothesis is that enzymes in the ? membrane catalyze the oxidation of NH+ 4 with NO2 , with hydrazine and hydroxylamine as intermediates, and a proton motive force is created across the membrane, which is used for ATP production. The very tight membrane structure limits the diffusion of protons across the membrane enhancing ATP production. It also prevents loss of the reaction intermediates, and it con?nes the very reactive intermediate, hydrazine, to the anammoxosome and thus prevents it from doing damage to the rest of the cell [38]. 2.2. Identity The ?rst discovered anammox bacterium belongs to the Planctomycetales [28] and, while not in pure culture, was named Candidatus Brocadia anammoxidans. Subsequently, waste water treatment plants have yielded other anammox bacteria, and to date three genera have been described: Brocadia, Kuenenia and Scalindua [26]. Within the genus Scalindua, two marine molecular isolates have been identi?ed. One of these, found in the anoxic water column of the Black Sea [15], has been given a tentative species designation S. sorokinii, and another has been found in the sediment of Randers Fjord [22]. In both these locations the anammox process was documented by the use of 15 N compound labelling experiments (see Section 3.1). In the Black Sea the occurrence of anammox bacteria was further substantiated by the presence of the unique ladderane lipids. Thus, the cell structural properties of anammox bacteria appear similar in waste water treatment plants and in nature. However, the occurrence of the anammox process and the simultaneous detection of Scalindua, does not rule out the possibility that other phylogenetically unrelated organisms may take part in the anaerobic oxidation of NH+ 4 in nature. 2.3. Growth of anammox bacteria Nothing is known about the growth rate of marine anammox bacteria, but the related bacteria from waste water treatment facilities grow slowly with doubling times of around 9 days under optimal conditions [29]. The optimal temperature for the waste water treatment organisms is 37 ? C [14], and one might expect even slower growth for anammox bacteria living at much lower temperatures as generally encountered in the marine environment. However, the temperature optima for marine anammox populations appear to conform to the local environment. For example, in the permanently

cold sediment of Young Sound, Greenland, with a temperature of < ?1 ? C year round, the temperature optimum for anammox was 12 ? C [25], and in the Skagerrak, with annual temperatures between 4 and 6 ? C, the optimum temperature was 15 ? C [7]. We await the isolation or enrichment of marine anammox bacteria for a better understanding of their growth characteristics. 3. Characteristics of marine anammox 3.1. Documentation and stoichiometry The discovery of anammox in marine sediments, and the subsequent identi?cation of anammox in various marine environments, was based on experiments using a 15 N isotope labelling technique [33]. These experiments are typically performed with anoxic homogenized sediment or anoxic water, and a preincubation is employed to consume ? any O2 , and, in some cases, also the NO? 3 or NO2 initially present. Three different amendments are then made in par15 NH+ + 14 NO? (B), and 15 NO? (C). allel: 15 NH+ 4 (A), 4 3 3 After a suitable incubation time, the N2 gas is extracted and analyzed on a mass spectrometer for the speci?c labelling of 15 N in N2 gas (14 N15 N and 15 N15 N). The production of 15 N labelled N in amendment A would require the oxi2 ? ? dation of NH+ 4 with oxidants other than O2 , NO3 or NO2 , which are not found in the incubation. Such a production has thus far not been observed (e.g., [32,33]). Any production of 14 N15 N in amendment B must thus be due to coupling 14 N in NO? and that is eviof the 15 N in NH+ 4 with the 3 + dence of anaerobic NH4 oxidation. Since NO? 3 is readily reduced to NO? in anoxic marine sediments and waters (see 2 ? ? below) it may be either NO3 or NO2 which is the direct oxidant of NH+ 4 . Having identi?ed the anaerobic oxidation of ? ? with NO NH+ 4 3 or NO2 in amendment B, the relative importance of this process for N2 production, as well as that of denitri?cation, are estimated with amendment C. Thus, in amendment C the 15 N labelling of the NO? 3 pool is very high, and denitri?cation will mostly produce N2 gas labelled 15 N15 N, combining 2 15 N atoms from the added 15 NO? . 3 ? Also, in amendment C the oxidation of NH+ 4 with NO3 or 14 N15 N by the combination of NO? 2 will mainly produce 14 N from NH+ and 15 N from NO? . There will sometimes, 4 3 however, be some native 14 N-labelled NO? 3 in amendment C incubations, but by knowing the exact 15 N labelling of the NO? 3 pool, the contribution of anaerobic ammonium oxidation and denitri?cation to N2 production can still be estimated [33]. The question now is how to identify the oxidant for ammonium. The reduction of NO? 3 to the oxidation state of N2 requires ?ve electrons, while three electrons are liberated in oxidizing NH+ 4 to N2 yielding a 5:3 stoichiometry for the reaction in Eq. (1).
+ + 3NO? 3 + 5NH4 → 4N2 + 9H2 O + 2H .

(1)

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With this stoichiometry, 1 out of 4 N2 molecules would be 15 15 formed solely from NH+ 4 which would give 25% N N la+ belling in amendment B (if no native NH4 is present). This labelling percentage, however, has not been observed. Al? ? ternatively, if NO? 3 were reduced to NO2 by NO3 -reducing ? bacteria in the environment, and if this NO2 was the oxidant of NH+ 4 , the oxidation state changes would be +3 and ?3 respectively and the stoichiometry would be 1:1 (Eq. (2)).
+ NO? 2 + NH4 → N2 + 2H2 O. 14 N15 N

(2)

This would explain the observed production stoichiometries as typically found with amendment B, and the + ability for NO? 2 to act as oxidant of NH4 was con?rmed ? + in experiments where only NO2 and NH4 were present [7]. The overall stoichiometry of anaerobic NH+ 4 oxidation in the marine environment is therefore the same as found for the anammox process operating during waste water treatment [35]. This indicates that the anammox process is responsible for anaerobic ammonia oxidation in the marine environment [6,7,22,25,33,34]. 3.2. Production of NO? 2 Nitrate is generally much more abundant in the marine environment than NO? 2 , and the anammox process must rely ? on other processes to reduce NO? 3 to NO2 . Yet, this may ? not limit the rate of anammox as NO3 is readily reduced to NO? 2 in suboxic marine environments. In the sediments of the Skagerrak (located between Denmark and Norway), the ? production of NO? 2 by NO3 reduction in anoxic sediment incubations was 4 times faster than NO? 2 consumption [7]. In similar incubations with arctic sediment, the NO? 2 production rate was also faster than its consumption rate [25], while in the Thames estuary, NO? 2 production balanced its consumption [34]. In Randers Fjord sediment anammox rates ? 15 derived from 15 NO? 3 and NO2 additions were similar, in? dicating that production of NO2 from NO? 3 was not limiting anammox [22]. Most published studies of anammox in marine environments have been based on anoxic incubations of homogenized sediment or water, with the NO? 2 originating from the [7,22,33,34]. The group of organreduction of added NO? 3 isms responsible for this reduction has not been identi?ed, although denitrifying bacteria, and those bacteria promot+ ing the dissimilatory reduction of NO? 3 to NH4 (DNRA), have NO? 2 as a free intermediate, and thus are likely candidates. The excretion of NO? 2 occurs, however, by many bacteria which have the ability to reduce NO? 2 further. It is at, or above, it’s limiting conprobably occurs when NO? 3 centration and nitrate reducers can cover all their electron acceptor needs with the NO? 3 . This is always the case dur15 ing the ?rst part of most N-amendment experiments with relatively large additions of NO? 3 . In this case, nitrate is in excess during the ?rst part of the incubation, and a large por? tion of it is reduced only to NO? 2 . The NO2 is then reduced
Fig. 1. Schematic representation of the distribution of selected dissolved species in the sediment porewater and the water above the sediment (A) and indication of the depth intervals in which selected processes occur (B). Concentrations are not drawn to scale.

during the later part of the experiment when NO? 3 is limiting (e.g., [7]). In natural sediments the situation may be similar, but ? with NO? 3 limitation occurring spatially. Thus, NO3 concentrations are highest in the upper part of the anoxic NO? 3containing zone of the sediment (Fig. 1), and NO? -utilizing 3 organisms here are likely NO? 3 saturated. Only in the lowest ? part of the NO? 3 zone does NO3 limitation probably occur. Nitrate reducers become NO? 3 limited at concentrations below 2–3 times their Km value for NO? 3 uptake. Assuming Km values on the order of 1 ?M, or even less [5], it ? would seem that NO? 3 reduction becomes NO3 limited be? low 2–3 ?M NO3 . In intact sediment from Randers Fjord ? ? (Denmark) the NO? 3 + NO2 ( NO3 ) concentration at the oxic–anoxic interface was around 20 ?M [22]. Only in the lowermost part of the NO? 3 zone was it at a limiting concentration. Therefore, in this sediment, NO? 3 reducers likely will release NO? for the anammox bacteria. Nitrite availability is 2 a prerequisite for anammox to be important in sediments in nature. 3.3. Kinetics The af?nity for NO? 2 uptake by anammox bacteria and denitrifying bacteria has been investigated in homogenized sediment from the Skagerrak. The ratio of anammox to denitri?cation did not vary as a function of NO? 2 concentration [7], and it was concluded that the af?nity for NO? 2 uptake was similar in the 2 groups of bacteria. Furthermore, the Km value for NO? 2 uptake was estimated to be below

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3 ?M, possibly as low as 0.1 ?M, for both processes in this sediment where anammox accounted for approximately 65% of the N2 production. The Km value for NH+ 4 uptake by marine anammox bacteria is dif?cult to investigate in the type of experiments used thus far to study sediment anammox. The problem is that + native NH+ 4 concentrations often are high, and NH4 production during continued mineralization ensures that NH+ 4 never reaches limiting levels. In Skagerrak sediments, increasing the NH+ 4 concentration from 50 to 250 ?M did not change the anammox rate, indicating that the Km value was well below 50 ?M [7]. In natural sediments and anoxic water columns, however, anammox may become NH+ 4 -limited. In sediments anammox may be physically separated from + the major NH+ 4 source, namely the release of NH4 from anaerobic mineralization deeper in the system. In situations like the one depicted in Fig. 1, NH+ 4 concentrations may become very low in part of the NO? 3 zone and may thus limit the anammox process. Alternatively, anammox bacteria may have to compete for NH+ 4 with organisms conducting nitri?cation or microphytobenthos (when light is available) assim+ ilating NH+ 4 [22]. In anoxic water columns NH4 limitation of anammox may occur as demonstrated in Golfo Dulce [6]. Here, NH+ 4 concentrations were close to the detection limit, and the addition of 10 ?M NH+ 4 stimulated the rate of anammox 2- to 4-fold. Thus, the in situ NH+ 4 concentration was at or below the Km value. 3.4. Effects of oxygen For anammox bacteria isolated from waste water treatment facilities, the anammox process appears to occur only under strictly anaerobic conditions. As little as 1.1 ?M O2 was suf?cient to completely inhibit anammox activity in a bioreactor [30]. The inhibition was reversible, and in experiments with intermittent aeration, the rate of anammox was the same before and after aeration [12]. There are no existing reports on the effects of O2 on anammox in the sea, but it must be expected that marine anammox is also inhibited by O2 . However, O2 concentrations likely ?uctuate in the NO? 3 -containing zones where marine anammox occurs and as a result, marine anammox may have developed a higher tolerance towards O2 . 4. Relative importance and regulation of marine anammox 4.1. Marine sediments The discovery of anammox in marine environments means that denitri?cation is no longer synonymous with the removal of ?xed nitrogen. The deep sediments (700 m) of the Skagerrak, which is part of the Danish belt seaway, support the maximum reported relative importance of anammox in N2 production [10,33]. Here, anammox was responsible

for 67–79% of the total N2 production. Thus, in this sediment, anammox was more important than denitri?cation in the removal of ?xed nitrogen. However, the relative importance of anammox in nitrogen removal is highly variable, and in some cases anammox activity has not been detected (Fig. 2A). The relative importance of anammox in sediments was originally correlated with sediment mineralization rate and water depth [33]. Thus, in the initial reports of anammox activity, anammox was responsible for 67, 24 and 2% of the N2 production in the sediment of Skagerrak S9 (700 m), Skagerrak S6 (380 m) and Aarhus Bay (16 m), respectively. Water depth and sediment mineralization are generally related because with a deeper water column a larger fraction of the organic matter is mineralized during transport to the sediment, and the organic loading of the sediment is thus lower. Although anammox was relatively more important at the deepest site of the Skagerrak, the absolute rates of anammox were 3–4 times higher at the two shallower sites (see Fig. 2). This pattern was also observed in a study of anammox and denitri?cation in 7 sediments [10] which ranged from the deep Skagerrak with a very low sediment mineralization rate, to the highly active sediment of Long Island Sound. Overall, a good negative correlation was observed between the relative importance of anammox and sediment mineralization rate. Also, similar to the original observations, anammox rates only varied by a factor of 6 between the stations, whereas, denitri?cation rates varied by a factor of 114, and was almost linearly dependent on the sediment mineralization rate. In the Thames estuary the relative importance of anammox in N2 formation was linearly correlated with sediment organic content [34]. Out of six stations, sampled along the estuary from the discharge area of two very big sewage treatment works in London, to the North Sea, only one site deviated from this pattern. The organic content decreased from the discharge area, where anammox was responsible for 8% of the N2 production, towards the sea, where anammox accounted for only 1% of the N2 production. Two explanations were offered for this trend. Since anammox was originally discovered in sewage treatment it was argued that the Thames is seeded by anammox bacteria from the sewage plant discharge. In this model the largest population of anammox bacteria is located closest to the discharge. Alternatively, the relationship between anammox and sediment organic content may arise because a higher organic content leads to a higher NO? 3 reduction which may lead ? to a higher NO2 release and thus more NO? 2 available for anammox [34]. In two other studies, the relative importance of anammox was linked to the availability of NO? 3 . Risgaard-Petersen et al. [22] compared 2 sites in 2 shallow Danish estuaries (<1 m deep) and found that anammox accounted for between 4 and 26% of the N2 production at the Randers Fjord site but was undetectable in Norsminde Fjord. They attributed this difference to different NO? 3 availability in

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Fig. 2. Relative anammox (anammox/total N2 production ×100%) (A) and absolute rates of anammox (B) and denitri?cation (C) measured in jar experiments with homogenized sediment. Rates and relative anammox are estimated from 15 NO? 3 addition experiments. The water depth, station name and reference are given on the x -axis. nd: not determined; bdl: below detection limit; ud: unpublished data. In Norsminde Fjord, Greece and Spain, only the relative contributions of the 2 processes was measured.

the sediment. In Randers Fjord, NO? 3 in the water column and nitri?cation rates in the sediment are high year round, and microsensor pro?les of porewater NO? 3 in the sediment showed that NO? penetrated into the anoxic part of 3 ? the sediment. In darkness, NO3 concentration decreased from 23 ?M at 1.4 mm depth (oxic–anoxic interface) to zero at a depth of 3.7 mm. In the light, O2 penetrated to a depth of 2.8 mm, where the NO? 3 concentration was 20 ?M, and NO? penetrated to 5.5 mm. In Norsminde 3 Fjord, however, NO? concentrations were low in the water 3 column in the summer months. Also, the sediment was covered with benthic microalgae which, through active uptake, further suppressed sediment NO? 3 concentrations. Indeed,

micropro?les showed that there was no measurable NO? 3 in the anoxic zone of the sediment. In continental shelf sediments from East and West Greenland, anammox was relatively important, ranging from 1 to 35% of the N2 production at 11 sites with water depths ranging from 36 to 100 m [25]. In this study, area-based rates of anammox activity were estimated from whole core incubations with 15 NO? 3 [23], and a striking linear correlation (R 2 = 0.96) between bottom water NO? 3 concentrations and area-based, absolute rates of anammox appeared. The NO? 3 concentrations in arctic bottom waters are rather stable over the year, so the NO? 3 concentration at the time of sampling is a good indicator of the general NO? 3 availability in those

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sediments [25]. Also, since these sediments are below the photic zone, there is no competition from microphytobenthos, as was the case in Norsminde Fjord [22]. Still, it is surprising that the anammox rates correlated so closely with the NO? 3 concentration in the bottom water. Sediment nitri?cation was signi?cant at all sites and the NO? 3 availability in the sediment was therefore not governed by the bottom water NO? 3 concentration alone [25]. The relation between relative anammox and sediment mineralization or water depth was not investigated as the former was not measured and the latter varied only between 36 and 100 m. Can we ?nd a common thread through these various observations? In Fig. 2 the published data on anammox in marine sediments have been ranked according to water depth, and it appears that the relative importance of anammox in N2 production and water depth do correlate, as originally suggested (Fig. 2A). The relationship is not perfect and other environmental variables may also play a role in this regulation. However, it is clear that the relative signi?cance of anammox in N2 production is highest at greater water depth. Even though a high contribution of anammox in N2 formation can occasionally be found at shallow depth, anammox is always relatively important for nitrogen removal at greater depths. If this trend continues, anammox would be responsible for at least 2/3 of the N2 production in sediments at depths greater than those in Fig. 2. The absolute rates of anammox, however, do not correlate with water depth, although there is a tendency towards higher rates in shallower water (Fig. 2B). Denitri?cation rates, on the other hand, do correlate with water depth, where the highest rates are found at shallow water depths and rates are always low for sediment in deep waters (Fig. 2C). In the sediments so far studied denitri?cation rates vary over a wider range (0.10–153 nmol cm?3 h?1 ) than anammox rates (0.08–11 nmol cm?3 h?1 ) and denitri?cation is thus apparently more responsive to organic carbon loading than anammox. One might imagine that high rates of NH+ 4 release in shallow water organic-rich sediments may stimulate anammox, but this is apparently not the case. One reason for this could be that anammox is already NH+ 4 -saturated in shallow sediments. It is also possible that the higher electron donor (organic matter) availability in organic rich sediments creates a higher demand for electron acceptor (i.e., NO? 2 and ? ), and that a smaller fraction of the reduced NO NO? 3 3 is lib? erated as NO2 . Anammox may thus not be able to keep up with denitri?cation when electron donor availability is high. These issues are obviously a ripe area for future research. As suggested in some of the studies mentioned above, anammox may also be regulated by the supply of NO? 3 from both bottom waters and from nitri?cation. Higher NO? 3 concentration in the bottom water or higher nitri?cation rates will result in a deeper penetration of NO? 3 into the sediment. Consequently, the zone where denitri?cation (and nitrate re? duction to ammonia) will be NO? 3 saturated, and where NO2 therefore may be excreted to the porewater (see above), will be thicker and anammox may become more important. In

situations where denitri?cation is electron-donor limited, an increase in the availability of organic matter would stimulate denitri?cation. The penetration of NO? 3 into the sediment would thus decrease and the thickness of the zone in which NO? 2 would be released, and available for anammox, would also decrease. Assuming that the rate of anammox per volume of sediment would remain constant, a thinner zone of anammox would give a lower overall rate of anammox in the sediment, and an even lower contribution of anammox to N2 production. Another important variable regulating the importance of anammox may be environmental stability. If anammox bacteria in nature grow as slowly as we believe (see above), then the anammox process is probably only signi?cant in stable environments where there is a prolonged time for the bacterial population to develop. Denitri?ers have much higher growth rates which gives them a competitive advantage over the anammox bacteria in ?uctuating environments. Clearly, the factors controlling the environmental significance of anammox are still poorly understood. The depth dependence in Fig. 2 may indicate that water depth, and therefore sediment mineralization rate, is the most important driver controlling the relative importance of anammox in N2 production. Other factors, then, such as NO? 3 concentration, microphytobenthos activity, seeding of anammox bacteria, or local gradients in organic content may provide secondary, though still important, control. 4.2. Anoxic water columns and sea ice The anammox process is also important in N2 production in anoxic water bodies. In Golfo Dulce, a restricted basin on the Paci?c coast of Costa Rica, anammox was found to account for 19–35% of the N2 formation in the anoxic bottom waters [6]. Nitrate-rich surface water from the Paci?c mixes into the anoxic bottom water of the basin, and the NH+ 4 produced by the mineralization of sinking organic matter in the water column was immediately consumed by anammox bacteria. As a result, the anoxic water + column was almost free of NH+ 4 , and the low NH4 concentrations limited the rate of anammox. In the anoxic water column denitri?cation was the main mineralization process, and the relative importance of anammox was thus determined by the relationship between NO? 3 consumption and NH+ production by the denitrifying community. Theoreti4 cally, anammox should be responsible for 29% of the N2 production provided all NH+ 4 production coupled to denitri?cation was oxidized by anammox [6]. However, this theoretical limit may be exceeded because of preferential degradation of nitrogen-rich organic matter by denitri?cation [19,37]. In the Black Sea, anammox was shown to occur in the anoxic, non-sul?dic, zone where the NH+ 4 from the ? anoxic bottom water meets the NO3 from the surface waters [15]. Thus, in this environment anammox is not closely coupled to denitri?cation and other stoichiometric constraints may apply.

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The anammox process has also been found active in arctic sea ice [24]. The process was not detectable in ice less than 1 year old, but it was found to account for up to 19% of the N2 production in individual layers of a several years old ice ?oe. However, anammox was only important in a thin horizon and was thus only responsible for 0–5% of the total nitrogen removal in the ice. It is argued that the anammox process, due to the very slow growth of the bacteria [13], requires stable conditions and a long time to develop, and this is why it was not present in the ?rst year ice.

increases in sediment organic loading apparently augment denitri?cation much more than anammox, and the latter is of much lower importance. It is suggested that other variables such as NO? 3 availability, local gradients in sediment organic content, or the presence of microphytobenthos, may regulate anammox within the overall frame de?ned by the organic loading of the sediment. The anammox process is also very important in anoxic water columns where it may account for 35% of the nitrogen removal. It is argued that anammox in sediments and anoxic water bodies may amount to 1/3–1/2 of the global marine nitrogen removal.

5. Anammox and the marine nitrogen budget Acknowledgements The removal of ?xed nitrogen from the marine environment is believed to occur predominantly in anoxic sediments and in suboxic waters including the oxygen minimum zones of the Arabian Sea and the north and south eastern equatorial Paci?c Ocean. By current estimates, sediments account for about 2/3 of the total N2 production, and the water column is responsible for the remaining 1/3 [4]. Sediments on the continental slope (>150 m depth), and in the deep sea are assumed to be responsible for 53% of the sediment N2 production [16]. If anammox is responsible for more than half of this (as current results thus far indicate), and furthermore, is responsible for a signi?cant part of the nitrogen removed at shallower depths, this “new” process may be expected to carry out 1/3–2/3 of the global sedimentary nitrogen removal. Considering that about 87% of the ocean is deeper than 1000 m and the average depth of the oceans is 3800 m [31] it may be expected that anammox will be the dominant nitrogen removal process in most sediments. Furthermore, if anammox is as important in the oxygen minimum zones of the oceans as in Golfo Dulce, it may be expected to account for 35% of the marine water column nitrogen removal. Anammox may thus be responsible for 1/3–1/2 of the global removal of ?xed nitrogen from the marine environment [9]. While a very coarse estimate, this number emphasizes the need for a more detailed mapping of anammox in both benthic and pelagic environments. T.D. was supported by the Swedish Foundation for Strategic Marine Research (MISTRA, Dnr 2001-108) and by the EU research project MedVeg (QLRT-2000-02456). We also acknowledge Financial support for ?eld work from the Danish Research Council. D.E.C. and B.T. were further supported by the Danish National Research Foundation.

References
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