The oxygen minimum zone (OMZ) of the Eastern Tropical South Pacific (ETSP) is 1 of the 3 major regions in the world where oceanic nitrogen is lost in the pelagic realm. The recent identification of anammox, instead of denitrification, as the likely prevalent pathway for nitrogen loss in this OMZ raises strong questions about our understanding of nitrogen cycling and organic matter remineralization in these waters. anammox ͉ dissimilatory nitrate reduction to ammonium ͉ nitrogen loss ͉ functional gene expression ͉ remineralization

Active expression of putative ammonia monooxygenase gene subunit A (amoA) of marine group I Crenarchaeota has been detected in the Black Sea water column. It reached its maximum, as quantified by reverse-transcription quantitative PCR, exactly at the nitrate maximum or the nitrification zone modeled in the lower oxic zone. Crenarchaeal amoA expression could explain 74.5% of the nitrite variations in the lower oxic zone. In comparison, amoA expression by ␥-proteobacterial ammonia-oxidizing bacteria (AOB) showed two distinct maxima, one in the modeled nitrification zone and one in the suboxic zone. Neither the amoA expression by crenarchaea nor that by ␤-proteobacterial AOB was significantly elevated in this latter zone. Nitrification in the suboxic zone, most likely microaerobic in nature, was verified by 15 ammonia-oxidizing bacteria ͉ amoA gene expression ͉ marine group ͉ Crenarchaeota ͉ marine nitrogen loss N itrification, the stepwise oxidation of ammonium to nitrite and then nitrate, is a key process in marine nitrogen cycling. It is responsible for the formation of the large deep-sea nitrate reservoir. It connects the recycling of organic nitrogen to the ultimate nitrogen loss from the oceans, because its products are substrates for denitrification and anaerobic ammonium oxidation (anammox), the only two presently known nitrogen loss processes. In productive waters such as upwelling regions, high fluxes of organic matter and thus remineralization create strong subsurface oxygen minima, enabling denitrification (1-4) or anammox (5-8) to occur. Nitrogen losses from these oxygen minimum zones (OMZs) are estimated to account for 30-50% of total nitrogen loss from the oceans (9, 10). Because remineralization also releases large amounts of ammonium, high nitrification rates are often associated with these OMZs (11), implying that nitrification may play an important role in promoting marine nitrogen loss.The Black Sea is the largest marine anoxic basin in the world. A 20-to 40-m-thick suboxic transitional zone, characterized by low oxygen (Ͻ5 M) and undetectable sulfide, persists throughout the basin between the surface oxic layer and the sulfidic anoxic deep water (Ն100 m) (12, 13). The exact depth zonation varies according to the location within the basin because of circulation and gyre formation, but similar concentrations of chemical species can be traced along isopycnals or density ( t ) surfaces throughout the basin (12). Therefore, the Black Sea provides an ideal model system to study nitrogen cycling processes along oxygen gradients. Nitrification has been reported in the lower oxic zone (14) and so has nitrogen loss via anammox in the suboxic zone (15). Nevertheless, the identity and abundance of the responsible nitrifiers, or any coupling between nitrification and nitrogen losses, remain poorly documented.The first and rate-limiting step of nitrification is aerobic ammonia oxidation. It is a microbially mediated reaction. For decades, only specific groups of ␤-and ␥-proteobacteria have been found to exh...

Nitrite oxidation is the second step of nitrification. It is the primary source of oceanic nitrate, the predominant form of bioavailable nitrogen in the ocean. Despite its obvious importance, nitrite oxidation has rarely been investigated in marine settings. We determined nitrite oxidation rates directly in 15 N-incubation experiments and compared the rates with those of nitrate reduction to nitrite, ammonia oxidation, anammox, denitrification, as well as dissimilatory nitrate/nitrite reduction to ammonium in the Namibian oxygen minimum zone (OMZ). Nitrite oxidation (p372 nM NO 2 À d À1 ) was detected throughout the OMZ even when in situ oxygen concentrations were low to non-detectable. Nitrite oxidation rates often exceeded ammonia oxidation rates, whereas nitrate reduction served as an alternative and significant source of nitrite. Nitrite oxidation and anammox co-occurred in these oxygen-deficient waters, suggesting that nitrite-oxidizing bacteria (NOB) likely compete with anammox bacteria for nitrite when substrate availability became low. Among all of the known NOB genera targeted via catalyzed reporter deposition fluorescence in situ hybridization, only Nitrospina and Nitrococcus were detectable in the Namibian OMZ samples investigated. These NOB were abundant throughout the OMZ and contributed up to B9% of total microbial community. Our combined results reveal that a considerable fraction of the recently recycled nitrogen or reduced NO 3 À was re-oxidized back to NO 3 À via nitrite oxidation, instead of being lost from the system through the anammox or denitrification pathways.

Nutrient measurements indicate that 30–50% of the total nitrogen (N) loss in the ocean occurs in oxygen minimum zones (OMZs). This pelagic N-removal takes place within only ∼0.1% of the ocean volume, hence moderate variations in the extent of OMZs due to global warming may have a large impact on the global N-cycle. We examined the effect of oxygen (O2) on anammox, NH3 oxidation and NO3 − reduction in 15N-labeling experiments with varying O2 concentrations (0–25 µmol L−1) in the Namibian and Peruvian OMZs. Our results show that O2 is a major controlling factor for anammox activity in OMZ waters. Based on our O2 assays we estimate the upper limit for anammox to be ∼20 µmol L−1. In contrast, NH3 oxidation to NO2 − and NO3 − reduction to NO2 − as the main NH4 + and NO2 − sources for anammox were only moderately affected by changing O2 concentrations. Intriguingly, aerobic NH3 oxidation was active at non-detectable concentrations of O2, while anaerobic NO3 − reduction was fully active up to at least 25 µmol L−1 O2. Hence, aerobic and anaerobic N-cycle pathways in OMZs can co-occur over a larger range of O2 concentrations than previously assumed. The zone where N-loss can occur is primarily controlled by the O2-sensitivity of anammox itself, and not by any effects of O2 on the tightly coupled pathways of aerobic NH3 oxidation and NO3 − reduction. With anammox bacteria in the marine environment being active at O2 levels ∼20 times higher than those known to inhibit their cultured counterparts, the oceanic volume potentially acting as a N-sink increases tenfold. The predicted expansion of OMZs may enlarge this volume even further. Our study provides the first robust estimates of O2 sensitivities for processes directly and indirectly connected with N-loss. These are essential to assess the effects of ocean de-oxygenation on oceanic N-cycling.

We investigated the pathways of N 2 production in the oxygen-deficient water column of the eastern tropical South Pacific off Iquique, Chile, at 20uS, through short anoxic incubations with 15 N-labelled nitrogen compounds. The location was characterized by steep chemical gradients, with oxygen decreasing to below detection at ,50-m depth, while nitrite reached 6 mmol L 21 and ammonium was less than 50 nmol L 21 . Ammonium was oxidized to N 2 with no lag phase during the incubations, and when only NH þ 4 was 15 N-labeled, 15 N appeared in the form of 14 N 15 N, whereas 15 N 15 N was not detected. Likewise, nitrite was reduced to N 2 at rates similar to the rates of ammonium oxidation, and when only NO 2 2 was 15 N-labeled, 15 N appeared mainly as 14 N 15 N, whereas 15 N 15 N appeared in only one incubation. These observations indicate that ammonium was oxidized and nitrite was reduced through the anammox reaction, whereas denitrification was generally not detected and, therefore, was a minor sink for nitrite. Anammox rates were highest, up to 0.7 nmol N 2 L 21 h 21 , just below the oxycline, whereas rates were undetectable, ,0.2 nmol N 2 L 21 h 21 , deeper in the oxygen-deficient zone. Instead of complete denitrification to N 2 , oxidation of organic matter during the incubations may have been coupled to reduction of nitrate to nitrite. This process was evident from strong increases in nitrite concentrations toward the end of the incubations. The results point to anammox as an active process in the major open-ocean oxygen-deficient zones, which are generally recognized as important sites of denitrification. Still, denitrification remains the simplest explanation for most of the nitrogen deficiency in these zones.

A combination of stable isotopes ( 15 N) and molecular ecological approaches was used to investigate the vertical distribution and mechanisms of biological N 2 production along a transect from the Omani coast to the central-northeastern (NE) Arabian Sea. The Arabian Sea harbors the thickest oxygen minimum zone (OMZ) in the world's oceans, and is considered to be a major site of oceanic nitrogen (N) loss. Short (o48 h) anoxic incubations with 15 N-labeled substrates and functional gene expression analyses showed that the anammox process was highly active, whereas denitrification was hardly detectable in the OMZ over the Omani shelf at least at the time of our sampling. Anammox was coupled with dissimilatory nitrite reduction to ammonium (DNRA), resulting in the production of double-15 N-labeled N 2 from 15 NO 2 À , a signal often taken as the lone evidence for denitrification in the past. Although the central-NE Arabian Sea has conventionally been regarded as the primary N-loss region, low potential N-loss rates at sporadic depths were detected at best. N-loss activities in this region likely experience high spatiotemporal variabilities as linked to the availability of organic matter. Our finding of greater N-loss associated with the more productive Omani upwelling region is consistent with results from other major OMZs. The close reliance of anammox on DNRA also highlights the need to take into account the effects of coupling N-transformations on oceanic N-loss and subsequent N-balance estimates.

We performed incubation experiments with 15 N-labeled nitrogen compounds to investigate the vertical distribution of pathways of N 2 production through the suboxic zone of the central Black Sea and the impact of oxygen and sulfide on the anammox process. Anammox rates increased with depth through the upper suboxic zone and reached a maximum of ,11 nmol N 2 L 21 d 21 at the sharp interface between nitrate and ammonium, below which rates decreased toward the depth of sulfide accumulation. Heterotrophic denitrification was not detected, and therefore anammox was the prevailing sink for fixed nitrogen in the central Black Sea. In incubations with low oxygen concentrations, anammox activity was only partially inhibited, with a decrease in anammox rates to ,70% and 50% of the anoxic level at ,3.5 and ,8 mmol L 21 O 2 , respectively, and complete inhibition at ,13.5 mmol L 21 O 2 . Thus, the anammox process is not constrained to anoxic marine waters. This increases the volume of the major open-ocean oxygen-deficient zones, where anammox is potentially active, which has important implications for the contribution of anammox to the marine nitrogen cycle. We observed an inhibitory effect of micromolar sulfide concentrations on anammox activity, indicating that the vertical and likely horizontal distribution of active anammox bacteria is constrained to nonsulfidic water layers, which may explain the absence of the process in sulfidic basins with no suboxic zone.The discovery of anaerobic ammonium oxidation (anammox) in wastewater treatment systems and natural aquatic environments resolved the mystery of ammonium deficiency in anoxic waters and challenged the preeminence of microbial denitrification as the only significant pathway for the removal of fixed nitrogen in the oceans (Devol 2003;Dalsgaard et al. 2005;Kuypers et al. 2006). Anaerobic ammonium oxidation with nitrite as an electron acceptor is mediated by a monophyletic group of bacteria that branches deeply in the phylum Planctomycetes (Strous et al. 1999;Schmid et al. 2003). The one-to-one coupling of nitrogen from ammonium and nitrite into gaseous N 2 , NH Graaf et al. 1995), distinguishes the anammox process from denitrification, where two molecules of nitrate are combined to N 2 in a stepwise pathway (2NOAlthough a number of studies demonstrate the importance of anammox bacteria in the biological nitrogen cycle (Dalsgaard et al. 2005;Kuypers et al. 2006;, little is known about the main factors that control the distribution and magnitude of the process. Expectedly, oxygen is such an important regulator. Experimental work with enrichments of anammox bacteria from laboratory wastewater bioreactors has shown that anammox activity is reversibly inhibited by oxygen levels as low as 1 mmol L 21 (Strous et al. 1997), indicating that the process is active only under strictly anoxic conditions. Still, in the Benguela upwelling system off Namibia, the observed dominance of anammox was suggested to result from anammox being less sensitive than denitrification tow...

Permeable or sandy sediments cover the majority of the seafloor on continental shelves worldwide, but little is known about their role in the coastal nitrogen cycle. We investigated the rates and controls of nitrogen loss at a sand flat (Janssand) in the central German Wadden Sea using multiple experimental approaches, including the nitrogen isotope pairing technique in intact core incubations, slurry incubations, a flow-through stirred retention reactor and microsensor measurements. Results indicate that permeable Janssand sediments are characterized by some of the highest potential denitrification rates (X0.19 mmol N m À2 h À1 ) in the marine environment. Moreover, several lines of evidence showed that denitrification occurred under oxic conditions. In intact cores, microsensor measurements showed that the zones of nitrate/nitrite and O 2 consumption overlapped. In slurry incubations conducted with 15 NO 3 À enrichment in gas-impermeable bags, denitrification assays revealed that N 2 production occurred at initial O 2 concentrations of up to B90 lM. Initial denitrification rates were not substantially affected by O 2 in surficial (0-4 cm) sediments, whereas rates increased by twofold with O 2 depletion in the at 4-6 cm depth interval. In a well mixed, flow-through stirred retention reactor (FTSRR), 29 N 2 and 30 N 2 were produced and O 2 was consumed simultaneously, as measured online using membrane inlet mass spectrometry. We hypothesize that the observed high denitrification rates in the presence of O 2 may result from the adaptation of denitrifying bacteria to recurrent tidally induced redox oscillations in permeable sediments at Janssand.