Anammox Bacteria: The Slow-Growing Autotrophs That Cut Aeration and Carbon Demand
Nitrogen removal by the conventional route is expensive twice over: you pay to oxidise ammonia all the way to nitrate, then you pay again in purchased carbon to reduce it back to nitrogen gas. Anammox bacteria short-circuit the whole cycle. Under anoxic conditions they use ammonia as the electron donor and nitrite as the electron acceptor and produce nitrogen gas (N2) directly, with no organic carbon and no aeration beyond the partial nitritation step.
Who they are
Every anammox bacterium found so far sits in the family Anammoxaceae within the order Planctomycetales, spread across six genera: Candidatus Brocadia, Candidatus Kuenenia, Candidatus Anammoxoglobus, Candidatus Jettenia, Candidatus Anammoximicrobium moscowii and Candidatus Scalindua. Candidatus Scalindua came out of the marine suboxic zone and is treated as the marine anammox lineage; the other five were all recovered from wastewater treatment systems and are grouped as freshwater anammox bacteria. They matter in two directions at once, as a major term in the global nitrogen cycle and as a working organism inside treatment plants.
How the reaction actually runs
Tracer work with 15N produced two candidate mechanisms, both built on the observation that oxidising N2H4 to N2 supplies the electrons needed to reduce NO2- to NH2OH. In one, a membrane-bound enzyme complex converts ammonia and NH2OH into N2H4, which is then oxidised to nitrogen gas in the periplasm, with the electrons passed back to reduce NO2- to NH2OH in the cytoplasm. In the other, N2H4 is formed in the cytoplasm and oxidised in the periplasm, with electrons travelling the transport chain to a cytoplasmic nitrite reductase (NIR). Both routes make hydroxylamine and hydrazine intermediates of the metabolism rather than side products, which is why they turn up in every mechanistic study.
The model that has stuck locates the key enzyme, hydrazine oxidoreductase (HZO), inside the anammoxosome. Hydrazine hydrolase (HH) converts NH4+ and hydroxylamine (NH2OH) into hydrazine; HZO, analogous to HAO in Nitrosomonas europaea, oxidises it inside the anammoxosome to N2, four protons and four electrons. Those four electrons, plus five protons drawn from the riboplasm, drive nitrite reduction to hydroxylamine through NIR. Because protons are consumed in the riboplasm and generated inside the anammoxosome, an electrochemical gradient builds across the anammoxosome membrane. That gradient stores chemical potential (Delta-pH) and electrical potential, together a proton motive force that pushes protons back out through the membrane-bound ATPase, which synthesises ATP and releases it into the riboplasm.
Enrichment and proof of function
Strous and co-workers characterised anammox sludge dominated by B. anammoxidans in an SBR. More than 70% of the enrichment was a single autotrophic organism. After fixing in a pH 7.4, 20 mmol/L K2HPO4/KH2PO4 buffer with 2.5% glutaraldehyde, the cells showed irregular morphology under electron microscopy, and an improved Percoll density gradient centrifugation gave a suspension with fewer than one heterotrophic bacterium per 200-800 cells. DNA extracted from that purified material was confirmed by PCR amplification and 16S rRNA analysis, establishing B. anammoxidans as the functional organism and placing anammox bacteria as a new autotrophic lineage within the Planctomycetales.
The genera differ in ways that matter to design. K. stuttgartiensis, recovered from biofilm reactors in Germany and Switzerland, behaves much like B. anammoxidans and looks similar under the electron microscope, but tolerates phosphate up to 20 mmol/L. Its optimum is pH 8.0 within a 6.5-9.0 working range, and 37 C; at 45 C activity disappears and does not return when the temperature comes back down, while at 11 C it holds roughly 24% of the 37 C rate, which pins it as a mesophilic, slightly alkaline organism. It reaches a maximum specific activity of 26.5 nmol/(mg.min) per unit protein, below B. anammoxidans, but stays active at lower cell density, with nitrite tolerance reported around 13 mmol/L. Scalindua sorokinii was found in the Black Sea and has since been split into S. brodae and S. wagneri. Anammoxoglobus propionicus can metabolise propionate but remains poorly characterised.
What has been demonstrated at scale
Performance depends heavily on reactor form. Dapena-Mora and colleagues measured a nitrogen loading rate of 2.0 g/(L.d) in an air-lift reactor with a maximum specific anammox activity (MSAA) of 0.9 g/(g.d); the same work in an SBR gave 0.75 g/(L.d) loading, an MSAA of 0.4 g/(g.d) and a NO2- removal rate of 99%. Jetten and co-workers ran the SHARON-ANAMMOX combined process on sludge digestion effluent, with the SHARON stage carrying a total nitrogen loading of 0.8 kg/(m3.d) and converting 53% of total nitrogen, 39% to NO2- and 14% to NO3-. Van Dongen and colleagues demonstrated long-term stable operation of SHARON-ANAMMOX at full plant scale, with nitrogen removal reaching 83% in the test.
Dijkman and Strous described CANON, in which oxygen is limited to below 0.5% air saturation so that aerobic ammonia-oxidising bacteria convert part of the NH4+ to nitrite and anammox bacteria finish the job in the same vessel. Later modelling work covered combined nitrification and anammox in biofilm, evaluating temperature and flow velocity for the process. Other groups have coupled the steps differently: methanogenesis to remove COD, partial oxidation to NO2-, then denitrification with NH4+ as the electron donor; or an EGSB reactor running at 6.56 g/(L.d) COD loading and 0.99 g/(L.d) nitrogen, returning 97% COD removal and 100% NO2- removal and achieving methanogenesis-anammox coupling in one vessel.