Nitrogen cycle

Solution to a marine mystery

The anammox reaction, a microbial process that was first observed in waste-water treatment plants, looks as if it may be a key player in the nitrogen cycle in certain parts of the oceans.

In the oceans, denitrification is the process by which nitrate (primarily) is reduced to N2 — inert dinitrogen gas. It occurs when certain bacteria decompose organic matter in environments where oxygen concentrations are vanishingly low, and is how 'fixed' nitrogen is converted into a form that cannot be used by most marine plants. This was believed to be the only mechanism of N2 production in the oceans — and so by far the largest marine sink for fixed nitrogen.

On pages 606 and 608 of this issue, however, Dalsgaard et al.1 and Kuypers et al.2 show that large-scale conversion of fixed nitrogen to N2 is probably occurring through another route. They find that N2 can be produced by the anaerobic oxidation of ammonia in the oceanic water column, and that this 'anammox reaction' may be common in natural marine environments. Although there was evidence that the anammox reaction occurs in marine sediments3, the new findings enlarge the picture and could significantly alter our understanding of nitrogen cycling in the ocean. Denitrification is balanced by nitrogen fixation, which is carried out in surface waters by highly specialized organisms that can reduce N2 for incorporation into organic tissue. The balance between the two processes is such that, over vast areas, plant productivity in the oceans is limited by the amount of fixed nitrogen4. So the details of denitrification and nitrogen fixation are very important.

In the upper, sunlit layer of the oceans, photosynthetic organisms produce organic matter in the proportion carbon:nitrogen:phosphorus of about 106:16:1. This is the Redfield ratio. When this organic matter sinks out of the sunlit layer and is decomposed, carbon dioxide, ammonia and phosphate increase in the same ratio. In oxygenated marine environments, the regenerated ammonia is oxidized to nitrate. But in oxygen-deficient environments, decomposition continues via denitrification, and nitrate concentrations begin to decrease while those of phosphate continue to increase. Oceanographers can estimate the amount of denitrification that has taken place from the difference between the amount of regenerated nitrate predicted from the measured phosphate concentration and the nitrate actually measured5. This calculation yields the amount of nitrate reduced to N2 during denitrification, and this decomposition should result in the 'remineralization' of ammonia — which, in the absence of oxygen, it was thought should remain as ammonia.

Many years ago, however, Richards6 pointed out that this ammonia could not be accounted for. He proposed that it might somehow be oxidized anaerobically to N2 gas by nitrite or nitrate, either inorganically or by some unknown microbe. Geochemical evidence that this reaction does indeed occur is strikingly clear in oxygen-deficient environments such as the Black Sea7. In 1999, an organism that could carry out this 'anammox' reaction was isolated from a bioreactor system designed to remove ammonia from waste water, and was identified as a relative of a group of bacteria called the planctomycetes8. But it was thought unlikely that these organisms were either common or of much importance in the environment9; and despite the geochemical evidence, and the existence of a suspect organism, this nitrogen-cycle pathway remained a mystery.

Using an elegant bit of sleuthing, the two groups1,2 have now found the smoking gun that shows that the anammox reaction can, indeed, proceed under environmental conditions in the oceans, and that the likely perpetrators are closely related to the organisms isolated from bioreactors. Both groups worked in suboxic areas where the geochemical distributions of nitrogen species suggested that the anammox process was occurring (see Fig. 1 of each paper on pages 606 and 609).

Dalsgaard et al.1 used isotope tracers of nitrogen to show that when radiolabelled ammonia (15NH3) was added to nitrate-containing water-column samples from the oxygen-depleted zone of an inlet on the west coast of Costa Rica, the 15N was incorporated into N2 when incubated in quasi-natural conditions. Furthermore, when both 15NH3 and isotopically labelled nitrate (15NO3) were added, the relative yield of 28N2, 29N2 and 30N2 showed that the anammox reaction followed the form

This conversion of ammonium and nitrite to N2 and water is the same as the anammox reaction seen in the waste-water bioreactor.

Similarly, Kuypers et al.2 show that 15NH3, added to samples from the oxygen-depleted zone in the Black Sea, was converted to N2 in the suboxic zone, substantiating the geochemical evidence7 that the anammox reaction is occurring there. To see if the anammox organism in the Black Sea is similar to the well-characterized planctomycete isolates, Kuypers et al. looked for the unique signature of strange lipids known as ladderanes. These lipids surround the anammoxosome, the unique, organelle-like structure within which the anammox reaction takes place. They found that the distribution of ladderanes in the water column closely mirrored the distribution of the anammox reaction as indicated by their nitrogen-isotope experiments. Furthermore, molecular DNA analyses showed that organisms closely related to the bioreactor planctomycetes were present and common at the relevant depths. Taken together, these studies1,2 present pretty compelling evidence that the anammox reaction takes place in the water column in natural marine environments, and that it is probably carried out by planctomycetes similar to those isolated from bioreactors.

So, just how important is this reaction in the oceans? Conditions favourable for denitrification occur primarily in the oxygen-deficient zones of water columns off the west coasts of Central and South America and India, and also in marine sediments, mainly on continental margins. The total amount is more or less equally partitioned between the water column and sediments4. Current estimates of water-column denitrification are based mostly on budget studies of nitrogen and phosphorus, and the Redfield ratio as outlined above. If anammox is responsible for the oxidation of regenerated ammonia, then 28% of the N2 production would be attributed to this reaction. If proteins are the preferential substrate for denitrification10, this percentage increases to 48%. Perhaps more importantly, sulphate reduction deeper in marine sediments produces large quantities of ammonia, which diffuse up into the denitrification zone where anammox could, again, rival denitrification as an N2-producing process.

All in all, it looks possible that anammox may account for between 30% and 50% of the N2 production in the oceans. If this is so, we will have to revise our ideas about the mechanisms of marine denitrification and how we think about the marine nitrogen budget.


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Correspondence to Allan H. Devol.

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