Anaerobic Ammonium Oxidation (ANAMMOX)

*The biological nitrogen cycle

-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.

*Anammox

-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.

*Initial research

-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).

*Anammox physiology

-Based on mass balances over anammox enrichment cultures, the anammox stoichiometry was estimated to be: 1 NH4+ + 1.32 NO2- +0.066 HCO3- + 0.13 H+ = 1.02 N2 + 0.26 NO3- + 0.066 CH2O0.5N0.15 + 2.03 H2O
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%>

*Identification of the bacterium

-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".

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