With the increased use of chemical fertilizers in agriculture, many densely populated countries face environmental problems associated with high ammonia emissions. The process of anaerobic ammonia oxidation ('anammox') is one of the most innovative technological advances in the removal of ammonia nitrogen from waste water. This new process combines ammonia and nitrite directly into dinitrogen gas. Until now, bacteria capable of anaerobically oxidizing ammonia had never been found and were known as "lithotrophs missing from nature". Here we report the discovery of this missing lithotroph and its identification as a new, autotrophic member of the order Planctomycetales, one of the major distinct divisions of the Bacteria. The new planctomycete grows extremely slowly, dividing only once every two weeks. At present, it cannot be cultivated by conventional microbiological techniques. The identification of this bacterium as the one responsible for anaerobic oxidation of ammonia makes an important contribution to the problem of unculturability.

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Until now, oxidation of ammonium has only been known to proceed under aerobic conditions. Recently, we observed that NH4+ was disappearing from a denitrifying fluidized bed reactor treating effluent from a methanogenic reactor. Both nitrate and ammonium consumption increased with concomitant gas production. A maximum ammonium removal rate of 0.4 kg N · m−3 · d−1 (1.2 mM/h) was observed. The evidence for this anaerobic ammonium oxidation was based on nitrogen and redox balances in continuous‐flow experiments. It was shown that for the oxidation of 5 mol ammonium, 3 mol nitrate were required, resulting in the formation of 4 mol dinitrogen gas. Subsequent batch experiments confirmed that the NH4+ conversion was nitrate dependent. It was concluded that anaerobic ammonium oxidation is a new process in which ammonium is oxidized with nitrate serving as the electron acceptor under anaerobic conditions, producing dinitrogen gas. This biological process has been given the name ‘Anammox” (anaerobic ammonium oxidation), and has been patented.

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Anaerobic ammonium oxidation (anammox) bacteria, which were discovered in waste-water sludge in the early 1990s, have the unique metabolic ability to combine ammonium and nitrite or nitrate to form nitrogen gas. This discovery led to the realization that a substantial part of the enormous nitrogen losses that are observed in the marine environment--up to 50% of the total nitrogen turnover--were due to the activity of these bacteria. In this Timeline, Gijs Kuenen recalls the discovery of these unique microorganisms and describes the continuing elucidation of their roles in environmental and industrial microbiology.