INNSENDTE ARTIKLER The Anammox process for nitrogen removal from wastewater – achievements and future challenges Av Beata Szatkowska and Bjarne Paulsrud Beata Szatkowska and Bjarne Paulsrud both work with Aquateam COWI AS. Sammendrag Summary Anaerob ammonium oksidasjon (Anammox) for Although the study of the Anammox process has fjerning av nitrogen fra ulike avløpsstrømmer er been ongoing for about 20 years and knowledge en relativt ny prosess som er blitt utviklet i løpet about Anammox bacteria has significantly increa- av de siste ca 20 år. Det gjenstår imidlertid mange sed, there are still many questions regarding uavklarte spørsmål og tilhørende FoU-oppgaver bacteria behaviour and process performance knyttet bl.a. til Anammox-bakterienes metabo- involving these bacteria. Therefore, Anammox lisme og hvordan disse bakteriene kan utnyttes bacteria still require further research. på best mulig måte i fullskala renseanlegg for Conventional wastewater nitrogen removal fjerning av nitrogen. Dette gjelder ikke minst systems require a lot of energy for nitrification, hvordan prosessen kan utvikles til å fjerne nitro- and often an external organic carbon source for gen fra vanlig kommunalt avløpsvann, og ikke denitrification. The application of the Anammox bare fra «sidestrømmer» med høyt ammonium- process (which significantly reduces the need for innhold og høy temperatur. energy and does not need organic matter to con- Sammenlignet med konvensjonelle metoder vert nitrogen to gas) in the main stream of a for fjerning av nitrogen fra kommunalt avløps- wastewater treatment plant would be a great vann (nitrifikasjon/denitrifikasjon) er Anam- alternative to save energy costs for aeration. mox-prosessen svært energibesparende, og det Moreover, the organic matter present in waste- er ingen behov for en karbonkilde, slik denitri- water could be regarded as a source of additional fikasjonsprosessen krever. Dette vil gi mulighe- energy. Such a sustainable approach is now ter for å ta ut så mye som mulig av det organiske intensely studied around the world. Its imple- materialet i avløpsvannet som primærslam og mentation seems to be a revolution in the field produsere fornybar energi av dette i form av bio- of sustainable wastewater treatment. This brings gass. Dersom man lykkes med å implementere a vision that soon the pollutants in the waste- Anammox-prosessen for fjerning av nitrogen fra water will no longer be seen as a problem, but as kommunalt avløpsvann, vil dette innebære en a source of renewable energy. kraftig økning av bærekraften til avløpsrense- anlegg, og det er ikke lenger utopisk å se på avløps- Introduction renseanlegg som en netto energiprodusent. Great efforts are made today in the field of nitro- gen removal from municipal wastewater, since 186 VANN I 02 2014 INNSENDTE ARTIKLER nitrogen is one of the compounds that are regu- The Anammox reaction lated in the discharge permit of Wastewater The overall reaction 1, half reactions 2, 3 and 4 Treat ment Plants (WWTPs) in many countries. (Kartal et al., 2011) and reaction 5 with cell synt- There are in the nature groups of bacteria which hesis proposed by Strous at al. (1998), are presen- are well known and have been widely applied in ted below: + - biological wastewater treatment systems. One of NH4 + NO2 = N2 + 2H2O (reaction 1) them, the nitrifiers, has the ability to oxidise - + - ammonium with oxygen into nitrite which is NO2 + 2H + e = NO + H2O (reaction 2) next oxidised into nitrates (nitrification). The + + - second group of bacteria, the denitrifiers, reduces NO + NH4 + + 2H + 3e = N2H4 + H2O oxidized forms of nitrogen in response to the oxi- (reaction 3) dation of an electron donor such as organic + - matter (denitrification). Both processes involve N2H4 = N2 + 4H + 4e (reaction 4) large expenditures: nitrification requires aeration, + - - + and denitrification needs supply of organic NH4 + 1.32NO2 + 0.066HCO3 + 0.13H → - matter if not sufficient in incoming wastewater. 1.02N2 + 0.26NO3 + 0.066CH2O0.5N0.15 + However, in the last few decades our under- 2.03H2O (reaction 5) standing of the nitrogen cycle on the earth has changed drastically, and therefore the concept of The first step involves the reduction of nitrite to biological wastewater treatment has evolved nitric oxide by nitrate reductase (reaction 2). Then rapidly. New and more sustainable solutions for ammonium is combined with nitric oxide by wastewater treatment have appeared, since more hydrazine hydrolase to the form of hydrazine effective and autotrophic bacteria responsible for (reaction 3). In the final step (reaction 4) hydrazine is the anaerobic ammonium oxidation (Anam- oxidised to dinitrogen gas via hydrazine/ mox) reaction has been discovered. Before these hydroxylamine oxidoreductase (Kartal et al., bacteria were identified by micro-biologists, the 2011). The Anammox reaction is always associated existence of Anammox bacteria was predicted in with nitrate production. These reactions occur 1977 by Engelbert Broda, who pointed out within the anammoxosome, a specialized pseudo- an aerobic ammonium oxidisers as “missing in organelle within the bacterium, and create a nature”, based on thermodynamic considera- proton gradient across the anammoxosome tions. membrane (van Niftrik et al., 2008). Research ers The first evidence of anaerobic ammonium propose different stoichiometrical quotients for oxidation to dinitrogen gas was obtained from a individual components of the Anammox reac- denitrifying fluidized-bed reactor system tions but always the same and important for the (Mulder et al., 1995). It was discovered that these process is nitrite to ammonium ratio (NAR) organisms related to Planctomycetales were which is equal to 1.3. Moreover, similarities are capable of oxidizing ammonium using nitrite visible in released nitrogen: per 2.3 moles of instead of O2 as the electron acceptor (Strous et ammonium and nitrite, about 1 mole of nitrogen al., 1999a). Moreover, it occurred that these gas (N2) is generated. From reaction 5 it can be microorganisms have a number of unique featu- seen that 0.066 mole of carbon is fixed per 1 mole res, including the use of hydrazine (N2H4, i.e., of oxidized ammonium, which equals to 1 carbon rocket fuel and normally poisonous to living per 15 catabolic cycles. Anammox bacteria are organisms) as a free catabolic intermediate. obligate anaerobic chemolithoautotrophs. The discovery of the Anammox bacteria changed the view of the nitrogen cycle and resul- Microbial characteristics ted in a remarkably active wastewater treatment Anammox bacteria have complicated and not yet research around the world. well understood microbiology (Kartal et al., VANN I 02 2014 187 INNSENDTE ARTIKLER 2012). Bacteria share numerous properties with major source of nitrogen release into the atmo- both eukaryotes and archaea. Particularly note- sphere from the oceans (Kartal et al., 2010). The worthy is the exceptional construction of a cell Anammox bacterial population was even found which is divided into three separate compart- in deep marine hypersaline gradient systems ments by bilayer membranes and consists of the (Borin et al., 2013). Moreover, it was detected in cell wall, paryphoplasm, riboplasm, and anam- terrestrial ecosystems as marshes, lakeshores, a moxosome. The knowledge about the composi- contaminated porous aquifer, permafrost soil, tion or function of both the cell wall and the agricultural soil and in samples associated with paryphoplasm compartment is limited. The nitrophilic or nitrogen-fixing plants (Humbert et Anammox reaction takes place in the anammo- al., 2010). It has even been estimated that about xosome which occupies most of the cell volume 50% of the annual fixed nitrogen loss on the earth and is a so-called “prokaryotic organelle” could be attributed to Anammox activity (Lam & (Lindsay et al., 2001). The current knowledge Kuypers, 2011). In the natural environment the about Anammox cell biology is presented by Anammox process has been reported to occur at Niftrik and Jetten (2012). temperatures as low as −2.5°C in sea ice (Dals- So far 10 Anammox species have been identi- gaard and Thamdrup, 2002; Rysgaard and Glud, fied. Known species are divided into 5 genera: 2004) and as high as 70°C in hot springs and (1) Kuenenia, represented by Kuenenia stutt- hydrothermal vent areas (Byrne et al., 2009, gartiensis; (2) Brocadia including 3 species: B. Jaeschke et al., 2009). anammoxidans, B. fulgida, and B. sinica; (3) Anammoxoglobus, 1 species: A. propionicus; (4) Metabolism inhibition Jettenia 1 species: J. asiatica; and (5) Scalindua As Anammox bacteria favour anaerobic condi- having 4 species: S. brodae, S. sorokinii, S. wag- tions, the Anammox process is reversibly inhibi- neri, and S. profunda (Kartal et al., 2013). ted by the dissolved oxygen (DO) concentration. Phylogenetic analysis places them all within the The effect of oxygen on process performance is phylum Planctomycete. described in the following sub-chapter. The effect All the five currently recognized genera of of oxygen on Anammox bacteria has been exa- Anammox bacteria share unique physiological mined in marine ecosystems (OMZs). The results and morphological features with the key one showed that oxygen is a major controling factor being the presence of the anammoxosome. for Anammox activity in OMZ waters (Kalvelage Nearly two thousands of these gene sequences of et al., 2011). 16S rRNA affiliated with Anammox bacteria Despite the fact that nitrite is a substrate for have been deposited in the GenBank