Anaerobic ammonium oxidation by anammox bacteria in the Black Sea

Abstract

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 world's 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.

Improved analysis of ladderane lipids in biomass and sediments using high‐performance liquid chromatography/atmospheric pressure chemical ionization tandem mass spectrometry

“…A qualitative standard mixture containing the heptyl-[3]-and - [5]-, and pentyl- [5]-ladderane FAMEs (described in Sinninghe Damsté et al 8 ) was available for method optimization. Quantitative standards of the fatty acid methyl esters of the heptyl- [3]-and heptyl- [5]-ladderanes, as well as the monoether, were prepared from a large batch of anammox bacterial biomass.…”

“…Quantitative standards of the fatty acid methyl esters of the heptyl- [3]-and heptyl- [5]-ladderanes, as well as the monoether, were prepared from a large batch of anammox bacterial biomass. 2 Specifically, freeze-dried culture material was ultrasonically extracted three times each with methanol (MeOH), MeOH/dichloromethane (DCM) (1:1, v/v) and DCM.…”

“…Detection was achieved using positive ion APCI and selective reaction monitoring (SRM). The following optimal source settings were determined using loop injections of a qualitative mixture of the heptyl-[3]-and - [5]-and pentyl- [5]ladderane FAMEs, and monoether: 8 vaporizer temperature 4758C, discharge current 2.5 mA, sheath gas (N 2 ) pressure 50 (arbitrary units), auxiliary gas (N 2 ) pressure 5 (arbitrary units), capillary temperature 3508C, source CID À10 V. The argon pressure was maintained at 1.5 mTorr in the second quadrupole. Optimal collision energies varied depending on the monitored reaction and were determined by automatic tuning.…”

“…This is required to maintain concentration gradients during the exceptionally slow anammox metabolism and to protect the remainder of the cell from toxic anammox intermediates such as hydrazine. Lipids containing the biochemically unique [5]-and [3]ladderane moieties (Fig. 1, structures A and B, respectively) exist in a variety of forms.…”

“…[3][4][5][6][7] Kuypers et al 5 were the first to identify ladderane lipids in a natural seawater sample using gas chromatography (GC) and GC/mass spectrometry (GC/MS) analysis. Using a combination of molecular biological techniques, 15 N-labelling studies and the ladderane lipids as unique biomarkers, they identified the anammox process in the water column of the Black Sea.…”