Anammox in the Treatment Plant: Cell Biology, Reactor Configurations and Reported Nitrogen Removal Rates

Anaerobic ammonium oxidation removes nitrogen without organic carbon and without the aeration bill that conventional nitrification-denitrification carries. The organisms responsible do it through a biochemistry unlike anything else in wastewater microbiology, and the reactor engineering has spent two decades catching up with that biology.

What the Organisms Are

Anammox bacteria are autotrophs that, under anoxic conditions, use ammonia as the electron donor and nitrite as the electron acceptor. All known species sit in the family Anammoxaceae within the order Planctomycetales, across six genera: Candidatus Brocadia, Candidatus Kuenenia, Candidatus Anammoxoglobus, Candidatus Jettenia, Candidatus Anammoximicrobium moscowii and Candidatus Scalindua. Only Candidatus Scalindua is found in the marine suboxic zone and called the marine anammox bacterium; the other five genera occur in wastewater treatment systems and are described as freshwater anammox bacteria. They matter both for the global nitrogen cycle and for wastewater treatment.

Cells are morphologically diverse - spherical, ovoid and so on - at 0.8-1.1 micrometre in diameter, and Gram-negative. There is no capsule outside the cell; the wall surface carries crater-like structures and a few cells have pili. The wall itself is built mainly from protein and contains no peptidoglycan at all.

A Cell Built Around a Reaction Vessel

Each cell divides into three parts: the anammoxosome, the riboplasm and the paryphoplasm. Ribosomes and the nucleoid sit in the riboplasm, which holds most of the DNA. The anammoxosome is a structure unique to anammox bacteria, accounting for 50%-80% of cell volume, and it is where the anammox reaction takes place; a double membrane surrounds it and dips deeply into it. Hydroxylamine and hydrazine are intermediates of the metabolic process, and the small, toxic hydrazine is generated inside the anammoxosome. Its membrane lipid lets the cell make use of the chemical energy while containing that toxicity.

That membrane is the striking part. It contains special ladderane lipids, formed from multiple cyclobutane units and shaped like a ladder, whose content is roughly similar across anammox bacteria. The hydrophobic ladderane lipids combine with hydrophilic phosphorylcholine, phosphoethanolamine or glycerophosphate to form the phospholipids making up the membrane skeleton. Non-ladderane membrane lipids are straight-chain fatty acids, branched fatty acids, monounsaturated fatty acids and triterpenes. Ladderanes were once thought to sit only on the anammoxosome double membrane, limiting diffusion of toxic intermediates; they are now believed to occur on all membrane structures including the cytoplasmic membrane, combined with non-ladderane lipids so that other membranes stay more permeable than the anammoxosome.

The Metabolic Model

Van de Graaf and colleagues used N as a tracer element to work out the pathway, on the assumption that conversion of N2H4 to N2 provides the electrons needed for the reaction reducing N02 to NH2OH. Two mechanisms were proposed. In the first, a membrane-bound enzyme complex converts ammonia and NH2OH into N2H4, which is oxidised to nitrogen in the periplasm, and the electrons produced are transferred internally to reduce N02 to NH2OH in the cytoplasm via an enzyme complex that also handles N2H4 oxidation. In the second, ammonia and NH2OH are converted to N2H4 in the cytoplasm by a membrane-bound enzyme complex, N2H4 is converted to N2 in the periplasm, and the electrons are passed along the electron transport chain to cytoplasmic nitrite reductase.

The version most often cited places the key enzyme, hydrazine oxidoreductase (HZO), inside the anammoxosome. Hydrazine hydrolase (HH) converts NH4 together with hydroxylamine (NH2OH) into hydrazine; HZO, which is similar to HAO in N. europaea, oxidises it, producing N2, four protons and four electrons inside the anammoxosome. Those four electrons, with five protons taken from the riboplasm, drive nitrite reductase (NIR) in reducing nitrite to hydroxylamine. Because protons are consumed in the riboplasm and generated inside the anammoxosome, an electrochemical gradient builds up across the membrane - part chemical potential, part electrical potential - and the resulting proton motive force pushes protons outward. ATP synthase bound to the anammoxosome membrane then synthesises adenosine triphosphate (ATP), releasing it into the riboplasm as protons migrate back through the enzyme's proton pore.

The Known Species and What They Tolerate

Strous and co-workers characterised anammox sludge dominated by B. anammoxidans in an SBR reactor, where more than 70% of the enriched bacteria were a single dominant autotroph. After treatment with a pH 7.4 buffer of 20 mmol/L K2HPO4/KH2PO4 and 2.5% glutaraldehyde, the cells showed irregular microbial characteristics under the electron microscope. Improved Percoll density-gradient centrifugation produced very pure cell suspensions - fewer than one foreign bacterium per 200-800 cells. DNA extracted from those purified cells, amplified by PCR and analysed by 16S rRNA, established B. anammoxidans as the functional microorganism and confirmed anammox bacteria as a new autotrophic member of the Planctomycetales sequence.

Candidatus Kuenenia stuttgartiensis, found in biofilm reactors at several wastewater treatment plants in Germany and Switzerland, acts similarly to B. anammoxidans and looks similar under electron microscopy. It differs in tolerance: phosphate to 20 mmol/L and nitrite to 13 mmol/L, and it stays active at lower cell density. Its maximum anammox activity per unit protein is 26.5 nmol/(mg.min), below that of B. anammoxidans. The pH range is 6.5-9.0 with an optimum of 8.0 and an optimum temperature of 37 degrees C. At 45 degrees C no activity is observed and it does not recover when the temperature returns to 37 degrees C; at 11 degrees C activity is about 24% of the 37 degrees C figure - a mesophilic, slightly alkaline preference. Other described species remain poorly studied: Candidatus Scalindua sorokinii from the Black Sea, further split into Candidatus Scalindua brodae and Candidatus Scalindua wagneri, and Candidatus Anammoxoglobus propionicus, which can metabolise propionate.

Reactor Configurations and Reported Rates

Process work has concentrated on coupling partial nitritation with anammox. Jetten and colleagues treated sludge digestion effluent with a combined SHARON-ANAMMOX process: the SHARON reactor ran at a total nitrogen load of 0.8 kg/(m.d), converting 53% of total nitrogen - 39% to NO2 and 14% to NO3 - and with that effluent feeding an anammox fluidised-bed reactor, NO2 was completely removed in the nitrite-limited reactor while NH4-N removal reached 83%. Van Dongen and co-workers applied the same combination for long-term stable operation at a plant.

Mulder and colleagues first discovered anammox in an anaerobic fluidized bed; Van de Graaf and Bock later established the process with nitrite as electron acceptor; Zheng Ping and co-workers studied the kinetic characteristics of anammox mixed cultures. Other configurations report higher rates. Fux Christian and colleagues ran pilot tests oxidising ammonia in a continuous stirred tank reactor with 58% of NH4-N converted to nitrite, then completing anammox in an SBR for a nitrogen removal rate of 2.4 kg/(m.d) and 90% nitrogen removal. Sliekers and colleagues found 8.9 kg/(m.d) in an airlift reactor, twenty times the laboratory rate. Dapena-Mora and co-workers reported an N load rate of 2.0 g/(L.d) in an airlift reactor with a maximum specific anammox activity (MSAA) of 0.9 g/(g.d), and in an SBR an N load rate of 0.75 g/(L.d), MSAA of 0.4 g/(g.d) and NO2 removal of 99%.

CANON, described by Dijkman and Strous, runs both groups in one oxygen-limited vessel - under 0.5% air saturation - obtaining a co-culture of aerobic and anaerobic ammonia-oxidizing bacteria. Aerobic ammonia oxidisers such as Nitrosomonas and Nitrosospira oxidise NH4 to nitrite, and anammox bacteria convert that to nitrogen gas; the process rests on the synergy of the two autotrophic groups. Studied in an SBR and a chemostat at a volumetric N load of 0.1 kg/(m.d), nitrogen removal was 92%. Under oxygen limitation at load rates suited to both groups, an SBR reached 0.3 kg/(m.d) with NH4 mainly converted to N2 (85%) and the rest to nitrate (15%), and an airlift reactor reached 1.5 kg/(m.d). Hao and colleagues developed a mathematical model of combined nitrification and anammox in a biofilm reactor, evaluating temperature and flow velocity for CANON.

Coupling with methanogenesis is also reported. Jetten and co-workers removed COD by sludge digestion methanogenesis, partially oxidised nitrogen to nitrite, then denitrified using NH4 as electron donor. Zhang used EGSB reactor technology with COD removal of 97%, NO2 removal of 100% and volumetric loads of 6.56 g/(L.d) for COD and 0.99 g/(L.d) for nitrogen, achieving coupling of methanization, denitrification and anammox in one reactor.