A missing link in the nitrogen cycle

The Black Sea is stratified due to the cooler and less saline Mediterranean water that fills the basin, producing a significant anoxic layer from 80m. Phytoplankton live and die in the upper oxic layer, transferring fixed nitrogen to the anoxic deep water. There is however, a discrepancy between the concentration of ammonium in the deep water and the concentration of inorganic nitrogen in the suboxic .

Kuypers et al (2003) set out to investigate whether anaerobic ammonia oxidation (anammox) was the reason for this discrepancy. If nitrate is converted to nitrite in the suboxic, the nitrite can be used as the electron donor to convert ammonium from the sediments to nitrogen gas and water – resulting in a net loss of inorganic nitrogen.

The author tested the hypothesis using various methods. Firstly, a nutrient depth profile was created using a CTD to give the authors a picture of where the anammox reactions might be occurring. The profile showed that nitrate peaked at 65m and nitrite at 80m, but are both absent past 90m. Oxygen was absent past 80m, ruling out aerobic nitrification. Water samples were also incubated from different depths with radioactively labelled (N¹⁵) ammonium and (N¹⁴) nitrite knowing that one mole of each would produce one mole nitrogen gas, confirming if anammox was occurring; it was.

Knowing that the bacteria Planctomycetes undergo the anammox reaction, using a membrane bound organelle largely composed of ladderane lipids, the authors did a lipid analysis of water samples to identify if these lipids were present, and at what depth. Three lipids were identified, and all at similar depths to where nitrate and nitrite disappear.

Next they extracted DNA from water samples where the lipids were at the highest concentration, and used molecular cloning methods to create a phylogenetic tree. This confirmed the bacteria isolated from the Black Sea were related to bacteria capable of anammox, and were 98% similar to bacteria sequenced from bioreactor (sewage treatment).

Molecular cloning allowed them to create a specific oligonucleotide probe, which be used identify Planctomycete using FISH microscopy.

Using a diffusion model they estimated that anaerobic ammonium oxidation was occurring the Black Sea suboxic at 0.007  µM day^-1. When compared to what was known for bioreactors, it estimated that 300-3000 anammox cells ml^-1 would have to be present for this amount of ammonia oxidation per day. Luckily having already created an oligonucleotide for the Planctomycete, the FISH probe was able to analyse water samples and showed that there was 1900 (±) 800 anammox cells per ml.

Based on the area of the basin, they went on to predict that 0.3Tg of inorganic nitrogen per year may be lost through anammox reactions – a significant amount considering 14Tg of fixed nitrogen of produced through photosynthesis.

The results presented here are really valuable for the nitrogen cycle in these environmental conditions, showing a net loss of nitrogen in basins with a suboxic zone. There could be similarities in fjords for example, which are also stratified in a similar way due to different water densities.

I thought the authors were thorough in their methodology, using this variety of methods and knowledge of the subject. They built up a picture of what was occurring by using different analyses, giving very conclusive and reliable results.

Reference:

Kuypers, M. M., Sliekers, A. O., Lavik, G., Schmid, M., Jørgensen, B. B., Kuenen, J. G., ... & Jetten, M. S. (2003). Anaerobic ammonium oxidation by anammox bacteria in the Black Sea. Nature, 422(6932), 608-611.