Gaute Lavik

Anaerobic ammonium oxidation by anammox bacteria in the Black Sea

The availability of fixed inorganic nitrogen (nitrate, nitrite and ammonium) limits primary productivity in many oceanic regions. The conversion of nitrate to N2 by heterotrophic bacteria (denitrification) is believed to be the only important sink for fixed inorganic nitrogen in the ocean. Here we provide evidence for bacteria that anaerobically oxidize ammonium with nitrite to N2 in the worlds largest anoxic basin, the Black Sea. Phylogenetic analysis of 16S ribosomal RNA gene sequences shows that these bacteria are related to members of the order Planctomycetales performing the anammox (anaerobic ammonium oxidation) process in ammonium-removing bioreactors. Nutrient profiles, fluorescently labelled RNA probes, 15N tracer experiments and the distribution of specific ‘ladderane’ membrane lipids indicate that ammonium diffusing upwards from the anoxic deep water is consumed by anammox bacteria below the oxic zone. This is the first time that anammox bacteria have been identified and directly linked to the removal of fixed inorganic nitrogen in the environment. The widespread occurrence of ammonium consumption in suboxic marine settings indicates that anammox might be important in the oceanic nitrogen cycle.

Revising the nitrogen cycle in the Peruvian oxygen minimum zone

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. Without detectable denitrification, it is unclear how NH4+ is remineralized from organic matter and sustains anammox or how secondary NO2− maxima arise within the OMZ. Here we show that in the ETSP-OMZ, anammox obtains 67% or more of NO2− from nitrate reduction, and 33% or less from aerobic ammonia oxidation, based on stable-isotope pairing experiments corroborated by functional gene expression analyses. Dissimilatory nitrate reduction to ammonium was detected in an open-ocean setting. It occurred throughout the OMZ and could satisfy a substantial part of the NH4+ requirement for anammox. The remaining NH4+ came from remineralization via nitrate reduction and probably from microaerobic respiration. Altogether, deep-sea NO3− accounted for only ≈50% of the nitrogen loss in the ETSP, rather than 100% as commonly assumed. Because oceanic OMZs seem to be expanding because of global climate change, it is increasingly imperative to incorporate the correct nitrogen-loss pathways in global biogeochemical models to predict more accurately how the nitrogen cycle in our future ocean may respond.

Linking crenarchaeal and bacterial nitrification to anammox in the Black Sea

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 γ-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 β-proteobacterial AOB was significantly elevated in this latter zone. Nitrification in the suboxic zone, most likely microaerobic in nature, was verified by 15NO2− and 15N15N production in 15NH4+ incubations with no measurable oxygen. It provided a direct local source of nitrite for anammox in the suboxic zone. Both ammonia-oxidizing crenarchaea and γ-proteobacterial AOB were important nitrifiers in the Black Sea and were likely coupled to anammox in indirect and direct manners respectively. Each process supplied about half of the nitrite required by anammox, based on 15N-incubation experiments and modeled calculations. Because anammox is a major nitrogen loss in marine suboxic waters, such nitrification–anammox coupling potentially occurring also in oceanic oxygen minimum zones would act as a short circuit connecting regenerated ammonium to direct nitrogen loss, thus reducing the presumed direct contribution from deep-sea nitrate.

Detoxification of sulphidic African shelf waters by blooming chemolithotrophs

Coastal waters support ∼90 per cent of global fisheries and are therefore an important food reserve for our planet. Eutrophication of these waters, due to human activity, leads to severe oxygen depletion and the episodic occurrence of hydrogen sulphide—toxic to multi-cellular life—with disastrous consequences for coastal ecosytems. Here we show that an area of ∼7,000 km2 of African shelf, covered by sulphidic water, was detoxified by blooming bacteria that oxidized the biologically harmful sulphide to environmentally harmless colloidal sulphur and sulphate. Combined chemical analyses, stoichiometric modelling, isotopic incubations, comparative 16S ribosomal RNA, functional gene sequence analyses and fluorescence in situ hybridization indicate that the detoxification proceeded by chemolithotrophic oxidation of sulphide with nitrate and was mainly catalysed by two discrete populations of γ- and ε-proteobacteria. Chemolithotrophic bacteria, accounting for ∼20 per cent of the bacterioplankton in sulphidic waters, created a buffer zone between the toxic sulphidic subsurface waters and the oxic surface waters, where fish and other nekton live. This is the first time that large-scale detoxification of sulphidic waters by chemolithotrophs has been observed in an open-ocean system. The data suggest that sulphide can be completely consumed by bacteria in the subsurface waters and, thus, can be overlooked by remote sensing or monitoring of shallow coastal waters. Consequently, sulphidic bottom waters on continental shelves may be more common than previously believed, and could therefore have an important but as yet neglected effect on benthic communities.

Doubling of marine dinitrogen-fixation rates based on direct measurements

Biological dinitrogen fixation provides the largest input of nitrogen to the oceans, therefore exerting important control on the oceans nitrogen inventory and primary productivity. Nitrogen-isotope data from ocean sediments suggest that the marine-nitrogen inventory has been balanced for the past 3,000 years (ref. 4). Producing a balanced marine-nitrogen budget based on direct measurements has proved difficult, however, with nitrogen loss exceeding the gain from dinitrogen fixation by approximately 200 Tg N yr−1 (refs 5, 6). Here we present data from the Atlantic Ocean and show that the most widely used method of measuring oceanic N2-fixation rates underestimates the contribution of N2-fixing microorganisms (diazotrophs) relative to a newly developed method. Using molecular techniques to quantify the abundance of specific clades of diazotrophs in parallel with rates of 15N2 incorporation into particulate organic matter, we suggest that the difference between N2-fixation rates measured with the established method and those measured with the new method can be related to the composition of the diazotrophic community. Our data show that in areas dominated by Trichodesmium, the established method underestimates N2-fixation rates by an average of 62%. We also find that the newly developed method yields N2-fixation rates more than six times higher than those from the established method when unicellular, symbiotic cyanobacteria and γ-proteobacteria dominate the diazotrophic community. On the basis of average areal rates measured over the Atlantic Ocean, we calculated basin-wide N2-fixation rates of 14 ± 1 Tg N yr−1 and 24 ±1 Tg N yr−1 for the established and new methods, respectively. If our findings can be extrapolated to other ocean basins, this suggests that the global marine N2-fixation rate derived from direct measurements may increase from 103 ± 8 Tg N yr−1 to 177 ± 8 Tg N yr−1, and that the contribution of N2 fixers other than Trichodesmium is much more significant than was previously thought.

Nitrite oxidation in the Namibian oxygen minimum zone

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 15N-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 (⩽372 nM NO2− d−1) 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 ∼9% of total microbial community. Our combined results reveal that a considerable fraction of the recently recycled nitrogen or reduced NO3− was re-oxidized back to NO3− via nitrite oxidation, instead of being lost from the system through the anammox or denitrification pathways.

Oxygen sensitivity of anammox and coupled N-cycle processes in oxygen minimum zones

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.

Aerobic denitrification in permeable Wadden Sea sediments

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 (⩾0.19 mmol N m−2 h−1) 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 O2 consumption overlapped. In slurry incubations conducted with 15NO3− enrichment in gas-impermeable bags, denitrification assays revealed that N2 production occurred at initial O2 concentrations of up to ∼90 μM. Initial denitrification rates were not substantially affected by O2 in surficial (0–4 cm) sediments, whereas rates increased by twofold with O2 depletion in the at 4–6 cm depth interval. In a well mixed, flow-through stirred retention reactor (FTSRR), 29N2 and 30N2 were produced and O2 was consumed simultaneously, as measured online using membrane inlet mass spectrometry. We hypothesize that the observed high denitrification rates in the presence of O2 may result from the adaptation of denitrifying bacteria to recurrent tidally induced redox oscillations in permeable sediments at Janssand.

Biological and chemical sulfide oxidation in a Beggiatoa inhabited marine sediment

The ecological niche of nitrate-storing Beggiatoa, and their contribution to the removal of sulfide were investigated in coastal sediment. With microsensors a clear suboxic zone of 2–10 cm thick was identified, where neither oxygen nor free sulfide was detectable. In this zone most of the Beggiatoa were found, where they oxidize sulfide with internally stored nitrate. The sulfide input into the suboxic zone was dominated by an upward sulfide flux from deeper sediment, whereas the local production in the suboxic zone was much smaller. Despite their abundance, the calculated sulfide-oxidizing capacity of the Beggiatoa could account for only a small fraction of the total sulfide removal in the sediment. Consequently, most of the sulfide flux into the suboxic layer must have been removed by chemical processes, mainly by precipitation with Fe2+ and oxidation by Fe(III), which was coupled with a pH increase. The free Fe2+ diffusing upwards was oxidized by Mn(IV), resulting in a strong pH decrease. The nitrate storage capacity allows Beggiatoa to migrate randomly up and down in anoxic sediments with an accumulated gliding distance of 4 m before running out of nitrate. We propose that the steep sulfide gradient and corresponding high sulfide flux, a typical characteristic of Beggiatoa habitats, is not needed for their metabolic performance, but rather used as a chemotactic cue by the highly motile filaments to avoid getting lost at depth in the sediment. Indeed sulfide is a repellant for Beggiatoa.

Heterotrophic organisms dominate nitrogen fixation in the South Pacific Gyre

Oceanic subtropical gyres are considered biological deserts because of the extremely low availability of nutrients and thus minimum productivities. The major source of nutrient nitrogen in these ecosystems is N2-fixation. The South Pacific Gyre (SPG) is the largest ocean gyre in the world, but measurements of N2-fixation therein, or identification of microorganisms involved, are scarce. In the 2006/2007 austral summer, we investigated nitrogen and carbon assimilation at 11 stations throughout the SPG. In the ultra-oligotrophic waters of the SPG, the chlorophyll maxima reached as deep as 200 m. Surface primary production seemed limited by nitrogen, as dissolved inorganic carbon uptake was stimulated upon additions of 15N-labeled ammonium and leucine in our incubation experiments. N2-fixation was detectable throughout the upper 200 m at most stations, with rates ranging from 0.001 to 0.19 nM N h−1. N2-fixation in the SPG may account for the production of 8–20% of global oceanic new nitrogen. Interestingly, comparable 15N2-fixation rates were measured under light and dark conditions. Meanwhile, phylogenetic analyses for the functional gene biomarker nifH and its transcripts could not detect any common photoautotrophic diazotrophs, such as, Trichodesmium, but a prevalence of γ-proteobacteria and the unicellular photoheterotrophic Group A cyanobacteria. The dominance of these likely heterotrophic diazotrophs was further verified by quantitative PCR. Hence, our combined results show that the ultra-oligotrophic SPG harbors a hitherto unknown heterotrophic diazotrophic community, clearly distinct from other oceanic gyres previously visited.