RESEARCH NEWS Anammox – A novel microbial process for ammonium removal N. Shivaraman and Geetha Shivaraman Anammox1,2, a process of anaerobic Recently7 it was discovered that anam- moxasome is believed to be surrounded ammonium oxidation, is an innovative mox makes a significant (up to 70%) by a bilayer membrane that consists of technological advancement in the removal contribution to nitrogen cycling in the unique ‘ladderane’ lipids5. of ammonia nitrogen in waste water. world’s oceans. Figure 1 shows the The structure and function of anam- This new process combines ammonia and involvement of anammox process in the mox lipids has been published recently10. nitrite directly into dinitrogen gas3. The biological nitrogen cycle including meta- Lipid membranes are essential to the anammox reaction can be represented as bolic pathway for anaerobic ammonium functioning of cells facilitating the exis- + - NH 4 + NO 2 = N2 + 2H2O. This reaction oxidation. tence of concentration gradients of ions is carried out by anammox bacteria be- Initially anammox research5 was fo- and metabolites. Microbial membrane longing to the planctomycete group. Bac- cused on the basic properties of the proc- lipids are known to contain three-, five-, teria capable of anaerobically oxidizing ess and on providing evidence for its six-, and even seven-membered aliphatic ammonia were not identified earlier and microbial nature and the principles of the rings, but four-membered aliphatic cyclo- were known as ‘lithotrophs missing from nitrogen and carbon metabolism. It butane rings have been reported for the 4. Subsequently, this missing lith- appears that the anammox process is first time, in the dominant membrane otroph was discovered1 and identified as based on energy conservation from an- lipids of two anammox bacteria. These a new autotrophic member of the order oxic ammonium oxidation with nitrite as lipids contain up to five linearly fused Planctomycetales. It appears that they the electron acceptor and hydrazine and cyclobutane moieties with cis ring junc- might be the most primitive group of hydroxylamine as the intermediates. tions. These ‘ladderane’ moieties, rigid bacteria, at the very root of the bacterial Carbon dioxide is used as the main car- and dense ladders of concatenated cyclo- tree. Two of the anammox bacteria have bon source for growth. One of the key butane rings, occur in the membrane of 5 been named provisionally : Candidatus enzymes of anaerobic ammonium oxida- the anammoxasome, the intracytoplasmic ‘Brocadia anammoxidans’ and Candida- tion is hydroxylamine oxidoreductase8. compartment, where anammox catabo- tus ‘Kuenenia stuttgartiensis’. The former Its importance is illustrated by the fact lism takes place. A membrane with lad- bacterium was responsible for anaerobic that it constitutes 10% of the total cell deranes in its core is highly impermeable oxidation of ammonia observed in the protein. It catalyses the oxidation of hy- to passive diffusion of chemicals. Such a Netherlands, while the latter organism drazine and hydroxylamine. The enzyme membrane is required to maintain con- has been shown to be responsible for has been located in a membrane-boun- centration gradients during the very slow ammonia oxidation anaerobically in sev- ded, ‘organelle’ named anammoxasome, anammox metabolism. It also protects eral wastewater treatment plants in Ger- in the cytoplasm of the anammox cells9. the remainder of the cell from toxic many and Switzerland. Interestingly the This ‘organelle’ appears to be the center anammox intermediates, viz. hydroxyl- two species of the anammox bacteria of anaerobic ammonium oxidation. It is amine and hydrazine. Hydrazine and which have been characterized are rarely quite likely that its function could be hydroxylamine were observed to diffuse found together in a single anammox reac- containment of hydrazine. The anam- in and out of anammox cells as free tor, they must have different, as-yet un- molecules3. The containment of these known, ecological niches5. chemicals inside the anammoxasome was It is well known that the biological considered impossible, since both com- nitrogen cycle plays an important part in pounds diffuse through biomembranes. the maintenance of nitrogen balance in The discovery10 of the unprecedented global biosphere. The conventional bio- molecular structure of the anammox logical nitrogen cycle involves microbial membrane lipids shows that the anam- fixation of nitrogen gas to ammonia sym- moxasome membrane is much less per- biotically and non-symbiotically which is meable than normal biomembranes subsequently converted to organic nitro- because of the presence of unique ‘lad- gen. The ammonia released from organi- derane’ lipids. From a bioenergetic per- cally bound nitrogen and from man-made spective, the energy loss associated with activities, is biologically oxidized aero- loss of one molecule of hydrazine from bically to nitrite and then to nitrate. The the anammox cell is equivalent to at least resultant nitrate and nitrite are reduced to 10 full catabolic cycles, leading to 50% nitrogen gas by a denitrifying group of decrease in biomass yield at 10% hydra- bacteria using some electron donors (or- zine loss. For these reasons, limitation of ganic or inorganic compounds). It has diffusion is very important for these bac- been indicated6 that anaerobic ammonia Figure 1. Involvement of anammox pro- teria which are slow growers. One of cess in biological nitrogen cycle including oxidation also could be a major biologi- metabolic pathway for anaerobic ammo- these lipids was identified as a sn-2-gly- cal activity to be included in the nitrogen nium oxidation (adapted from refs 3 and cerol monoether with a C20 alkyl chain cycle, thus necessitating its modification. 5). containing four rings. The second lipid CURRENT SCIENCE, VOL. 84, NO. 12, 25 JUNE 2003 1507 RESEARCH NEWS was found to be a methyl ester of a C20 dinitrogen gas via nitrite intermediate, fertilizer manufacture, certain synthetic fatty acid containing five rings. From an oxygen supply needs to be controlled. fiber plants, explosive and latex process- evolutionary perspective we may note Thus, under oxygen limitation (< 0.5% ing industry, low temperature and high that part of the ladderane tails is ether air saturation) a coculture of aerobic and temperature carbonization process and linked to the glycol backbone. As of anaerobic ammonium oxidizers has been ammonium nitrate manufacturing unit in now, only a minority of bacteria (either obtained. These cultures converted am- steel plants. thermophiles or sulphate reducers) have monia directly to dinitrogen gas with ni- The research findings on this unique ether-linked lipids. Ladderane lipids can trite as the intermediate. Application of group of anammox bacteria have not be employed as molecular labels for trac- this concept in wastewater treatment only brought to light the missing litho- ing anammox activity in natural ecosys- could lead to complete ammonia removal troph involved in the nitrogen cycle, but tems. They are promising building blocks in a single autotrophic reactor5. This also have given scope for developing a in opto-electronics. Since the ladderanes concept has been named ‘CANON’, low cost biotechnology for control of are difficult to be synthesized chemi- meaning ‘completely autrophic nitrogen ammonia nitrogen pollution. cally, presently the anammox process is removal over nitrite’, and referring to the the only natural source. way the two groups of microorganisms Due to stringent wastewater discharge interact performing two sequential reac- 1. Strous, M. et al., Nature, 1999, 400, limits for ammonia (50 mg/l NH –N) and tions simultaneously5. While studying a 446–449. 4 2. van de Graaf, A. A., Mulder, A., de Bruijn, nitrate (10 mg/l NO –N) for inland sur- two-stage biological treatment system for 3 P., Jetten, M. S. M., Robertson, L. A. and face waters, it is essential to remove them ammonium nitrate laden wastewater, it Kuenen, J. G., Appl. Environ. Microbiol., to the permissible level. Biotreatment, was observed that after nitrate removal in 1995, 61, 1246–1251. being cost effective, is normally adopted the first stage fixed bed anoxic denitrify- 3. van de Graaf, A. A., de Bruijn, P., Rob- for their removal in wastewater. Conven- ing unit, there was substantial reduction ertson, L. A., Jetten, M. S. M. and Kue- tional biotreatment for ammonia removal in ammonia in a second stage pond sys- nen, J. G., Microbiology, 1997, 143, involves nitrification and denitrification. tem. Gas bubbles were seen generated 2415–2421. Biological nitrification is generally car- from the bottom of the pond. A possible 4. Broda, E., Z. Allgem. Mikrobiol., 1977, ried out by autotrophic nitrifying bacteria mechanism for ammonia removal has 17, 491–493. that oxidize ammonia to nitrite and been suggested analogous to the above 5. http://www.anammox.com/research.html. 6. Kuenen, J. G. and Jetten, M. S. M., ASM nitrite to nitrate with molecular oxygen CANON concept11. News, 2001, 67, 456–463. as electron acceptor. Nitrite and nitrate Very recently, a sequencing batch re- 7. Thamdrup, B. and Dalsgaard, T., Appl. are subsequently reduced to nitrogen gas actor (SBR) has been evaluated using a Environ. Microbiol., 2002, 68, 1312– by denitrifying bacteria, under anoxic mix of anammox bacteria and aerobic ni- 1318. conditions. However, this conventional trifying bacteria (predominantly bacteria 8. Schalk, J., De Vries, S., Kuenen, J. G. biological nitrification/denitrification pro- oxidizing ammonia to nitrite). In order to and Jetten, M. S. M., Biochemistry, cess is associated with the following obtain the right blend of these bacteria, 2000, 39, 5405–5412. requirements. Nitrification process de- the reactor was initially started anoxi- 9. Lindsay, M. R., Webb, R. I., Strous, M., mands very efficient oxygen supply cou- cally after inoculating with anammox Jetten, M. S. M., Butler, M. K., Forde, pled with adjustment of alkalinity of the bacteria. Subsequently oxygen was sup- R. J. and Fuerst, J. A., Arch. Microbiol., 2001, 175, 413–429. wastewater due to the formation of plied to the reactor and a nitrifying popu- 10. Damste, J. S. S.