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The biological nitrogen cycle plays an important part in the maintenance of the global biosphere. It has been a focus of microbiological investigations since the late nineteenth century. In the past 100 years, applied interests in the nitrogen cycle have shifted from improving agricultural crop yields to concerns about surface water pollution, destruction of the ozone layer and global warming.
 

Anaerobic ammonium oxidation (anammox), i.e. the microbiological conversion of ammonium and nitrite to dinitrogen gas, is a very recent addition to our understanding of the biological nitrogen cycle. Discovered as late as 1986, it so far is the most unexplored part of the cycle. Given its basic features, the anammox process is a viable option for biological wastewater treatment. Very recently, it was discovered that anammox makes a significant (up to 70%) contribution to nitrogen cycling in the World's oceans.
 

The first few years of anammox research have focused on the basic properties of the process and on providing evidence for its microbial nature and the principles of the nitrogen and carbon metabolism. It appeared that the anammox process is based on energy conservation from anoxic ammonium oxidation with nitrite as the electron acceptor and hydrazine as the intermediate. CO₂ is used as the main carbon source for growth. Recently we learned that CO₂ fixation is accomplished via the acetyl-CoA pathway. The necessary electrons are obtained from the anaerobic oxidation of nitrite to nitrate (see mechanism image to the right).

Based on mass balances over anammox enrichment cultures, the anammox stoichiometry was estimated to be:

Anammox is known to be active at temperatures between 6 and 43 degrees C. The pH range is 6.7 - 8.3 (optimum 8). Under optimum conditions, the maximum specific ammonium consumption rate is 55 micromol NH4+/g protein/min. The affinity for the substrates ammonium and nitrite is very high (affinity constants below 10 uM). Ammonia (100 mM) and nitrate (100 mM) do not inhibit the anammox process. The process is inhibited by nitrite concentrations higher than 20 mM. When the nitrite concentration is more than 5 mM for a longer period (12 h), anammox activity is completely lost. However, it can be restored by addition of trace amounts (50 uM) of either of hydrazine.

Anammox is inhibited completely at oxygen concentrations as low as 0.5% air saturation. Under oxygen limitation (<0.5% air saturation), a coculture of aerobic and anaerobic ammonium oxidizers can be obtained. This culture converts ammonium directly to dinitrogen gas, with nitrite as the intermediate. Application of this concept in wastewater treatment can lead to complete ammonia removal in a single, autotrophic reactor. This concept has been named CANON, meaning "completely autotrophic nitrogen removal over nitrite", and referring to the way the two groups of microorganisms interact: performing two sequential reactions simultaneously.

Mild sonication and density gradient centrifugation can be used to physically isolate the bacterium responsible for the anammox process in accordance with Koch's postulates. Anammox activity (18 micromol NH4+/g protein/min) of the isolated cells is dependent on cell density and on the addition of a trace amount (50 uM) of hydrazine. It was hypothesized that leakage of hydrazine out of anammox cells via passive diffusion is the explanation of cell density dependency of anammox cells. This has not yet been confirmed experimentally. The 16S rDNA sequence of the isolated bacterium was analyzed phylogenetically and grouped deep inside the Order Planctomycetales. Thus the microorganism responsible for anammox was identified as a new deep-branching planctomycete. It was named Candidatus "Brocadia anammoxidans".

The planctomycetes are an interesting group of bacteria with many rare or unique properties. They lack the otherwise universal bacterial cell wall polymer peptidoglycan, they divide by budding, and they have a differentiated cytoplasm, with different membrane-bounded compartments apparently allocated to different cellular functions. They are separated from other bacteria and amongst themselves by large evolutionary distances. Other species of planctomycetes are aerobic chemoorganoheterotrophs, very different from the anammox bacteria (which are anaerobic chemolithoautotrophs).

One of the key enzymes of anaerobic ammonium oxidation, hydroxylamine oxidoreductase was purified from Candidatus "B. anammoxidans". Its importance in anammox is illustrated by the fact that it made up 10% of the total cell protein. It catalyzed the oxidation of both hydrazine and hydroxylamine. This enzyme was located exclusively in a membrane-bounded, organelle-like body present in the cytoplasm of anammox cells. This 'organelle' was named the 'anammoxosome' and appears to be the locus of anaerobic ammonium oxidation by anammox cells. As yet it is unclear why anammox cells need an anammoxosome. It has been postulated that its function might be the containment of hydrazine or maintenance of the proton motive force at low proton translocation rates. Intact anammoxosomes have been purified from anammox cells (see Figure)

The anammoxosome is surrounded by a bilayer membrane that consists of unique and bizarre lipids. The anammoxosome lipids contain 'ladderane' moieties, rigid and dense ladders of concatenated cyclobutane rings. Both experimental evidence and molecular modelling have shown that a membrane with ladderanes in its core is extremely impermeable towards passive diffusion of chemicals. From an evolutionary perspective it is interesting to note that part of the ladderane tails are ether-linked to the glycerol backbone; up to now, only a minority of bacteria (either thermophiles or sulfate reducers) have ether linked lipids. Ladderane lipids are used as biomarkers for past and present anammox activity in natural ecosystems. Also, it appears that ladderane molecules have a future in high-tech applications such as opto-electronics. The anammox process is the only known natural source so far, and ladderanes are very difficult to produce synthetically.

Currently, at least three genera of anammox bacteria are known: Brocadia, Kuenenia and Scalindua. The first two have been found in wastewater treatment systems. The latter, Scalindua, has also been detected in marine ecosystems, such as the Black Sea and the benguella upwelling. The three genera share a common ancestor, but are evolutionally quite far apart (less than 85% sequence similarity on the 16S level). Still, all anammox bacteria seem to be very similar phenotypically: they all grow at the same, very slow rate, they all have an anammoxosome and ladderane lipids. The differences that must exist between the three genera are the topic of ongoing research.

In marine ecosystems, anammox bacteria actively contribute to biological nitrogen cycling, being responsible for at least 50% of total nitrogen production in the oceans. The anammox genus detected in the Black Sea is Candidatus "Scalindua sorokinii". Almost all marine systems investigated for anammox activity tested positive for this bacterium or close relatives.

The genome of Candidatus "K. stuttgartiensis" has been sequenced in a joint environmental genomics program of Genoscope, University of Vienna, the University of Nijmegen and the University of Munich. In this project, DNA was extracted from a complex bioreactor community dominated by Kuenenia stuttgartiensis. Over one billion base pairs were sequenced, the largest metagenome after Craig Venter's Sargasso sea. From these data the 4.3 Mb large genome of K. stuttgartiensis was pieced together. The genome exposed the genetic blueprint of hydrazine metabolism, ladderane biosynthesis and carbon fixation by anammox bacteria (via the acetyl-CoA pathway). It was also found that the planctomycetes and anammox bacteria in particular are related to a clade of intracellular parasites known as the chlamydiae.