Anaerobic ammonium oxidation (Anammox)

Denitrification and nitrification processes were found in 1882 and 1890, respectively (Fig. 4)[4].

Over 100 years after its identification of the denitrification process, the nitrogen cycle was generally believed to be completed.

Fig. 4 Timelines of discoveries in the fields of ammonium and Anammox [4].

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In 1995, Mulder et al. start up a 23-L capacity fluidized bed reactor for treating bakery yeast wastewater effluent in The Netherland [2]. Mulder et al. found that the nitrate and ammonium disappear at the same time in the reactor. The nitrification and denitrification could not remove ammonium only in an anoxic condition except assimilation. Excluding the assimilation, ammonium disappeared in the reactor, which raised interest by the researchers to advance survey on it. They recalled an article published in 1977 by Broda et al. which predicted that there are two lithotroph missing in nature [9]. One lithotroph is as reported to utilize ammonium as electron donor and nitrite as electron acceptor to form nitrogen gas. The difference between Mulder et al.

and Broda et al. is the electron donor form of utilized in the reaction. Eq. 9 represents the finding of Mulder et al.

5NH4+

+ 3NO3

 4N2 + 9H2O + 2H⁺ (9)

In eq. (9), the researcher considered that nitrate is used as the electron acceptor instead of nitrite.

Later, Graaf et al. demonstrated the utilization of nitrite as the electron acceptor by tracer experiment [10]. They designed a fluidized bed reactor introducing ¹⁵NH4+

and ¹⁴NO2

as tracers.

The end product of the reaction was nitrogen gas composed by ^14-15N₂. Two nitrogen atoms came from ¹⁵NH4+

and ¹⁴NO2

-. Graaf et al-. successfully demonstrated the anaerobic ammonium oxidation using nitrite and not by nitrate. In the Mulder et al. study [2], nitrate reduced to nitrite as the first step and combined ammonium and nitrite to proceed Anammox. Based on the amazing discovery, the nitrogen cycle in biological treatment was revised as shown in Fig. 5.

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Fig. 5 Biological nitrogen transformation cycle and Anammox shortcut.

Subsequently, Stous et al. formulated a complete metabolic equation for Anammox reaction based on the mass balance [11]. The protein content and elemental composition of the biomass were found to be CH2O0.5N0.15. According the biomass composition and mass balance, the stoichiometry of Anammox is illustrated in eq. (10).

NH4+

+1.32NO2

-+0.066HCO3

-+0.13H⁺  1.02N2+0.26NO3

-+0.066 CH2O0.5N0.15+2.03H2O (10)

Several interesting details can be noticed from the stoichiometry of Anammox. First, the biomass yield is extremely low, one mole of ammonium only yield 0.066 mole of the biomass. The extremely low yield of the biomass means the cultivation should spend a long time and also less sludge production saves sludge treatment cost. Second, Inorganic carbon is carbon source in the

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Anammox reaction. It is a kind of carbon fixation which could avoid global warming. Third, nitrite is the electron donor and also the electron acceptor. Nitrite reduces to ammonium as electron acceptor and oxidizes to nitrate to provide energy for assimilation biomass.

Since the Anammox bacteria growth rate is very low, researchers investigated the Anammox bacteria doubling time to evaluate the performance of Anammox bacteria growth [11-15]. The doubling time could be assessed by measuring the end product, i.e. nitrogen gas. When production of nitrogen gas is twice than the initial value, the period could be considered as doubling time. The first present the doubling time was 30 days [12]. Following the cultivation improved, the doubling time was shorted from 30 days to 11 days [11].

The bacteria, which found in the fluidized bed reactor in The Netherlands, were confirmed as a species of planctomycete. Planctomycete has been supposed only by a few organtrophs. Formerly the planctomycetes were considered to be of limited environmental importance. But this view changed as a molecular microbial ecology repeatedly providing new evidence that these bacteria are ubiquitous. Planctomycetes have the single- or double-membrane-bounded compartments separating their chromosome in the cytoplasm. They lack of peptidoglycan in their cell wall and are insensitive to ampicillin [16].

To seek out the origin of Anammox bacteria in the biggest anoxic basin, the researchers sampled sea water at different depth from the “Black Sea” [17]. They found that Anammox bacteria grow abundantly at the depth of 90 meters in sea. This evidence proved scientists the conversion of nitrite to nitrogen gas by heterotrophic bacteria and not by the major dinitrogen mechanism. From the sea water samples (depth of 100 meters), the 16S rRNA gene sequences were performed and the phylogenetic analysis was carried out. The results showed the Anammox bacteria are related to member of order Planctomycetales. The Anammox contributes at least 30% of the total nitrogen turnover in this inland sea. Anammox bacteria are abundant and important in the

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nitrogen cycle of the Black Sea. The species found in the Black Sea were identified as Candidatus Scalindua sorokinii [17]. The mystery of missing nitrogen in the ocean was solved by

investigation in the “Black Sea”. Anammox activity is also responsible for a major part of global nitrogen turnover [18].

The first discovered and identified Anammox bacteria is Candidatus Brocadia anammoxoidans, which found in a wastewater treatment plant [11]. Consequently, more Anammox bacteria were found in the wastewater treatments plants including Candidatus Kuenenia stuttgartiensis [19], Candidatus Scalindua brodae [20], Candidatus Scalindua wagneri [20], Candidatus

Anammoxoglobus propionius [21], Candidatus Brocadia [22], and Candidatus Jettenia asiatica [23].

A comparison of nitrification and Anammox is given in Table 1 [13, 24]. The major differences between nitrification and Anammox are the maximum specific ammonium consumption rate and growth rate. The ammonium consumption rate of Anammox is 22.5 times more than nitrification, but 1/100 less growth rate than nitrification. Furthermore, the Anammox cannot tolerate nitrite concentration over 20 mM for 12 hours.

The optimum Anammox growth temperature is between 20-43^oC. For example, a rotating biological contactor (RBC) handling Anammox was successfully operated at temperature 20^oC [25]. This result was similar to Isaka et al. where they operated an anaerobic biological filtrated (ABF) reactor for Anammox [26]. Moreover, many researchers analyzed the marine Anammox samples, and reported reasonably measurable activities at low temperature. Dosta et al. indicated that the maximum activity of Anammox was found at 35-40^oC; but if the temperature higher than 45^oC, an irreversible loss of the activity was observed due to the biomass lysis [24].

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Table 1 Comparison of nitrification and Anammox

Hydrazine and hydroxylamine were the two most important intermediates of Anammox [13, 27].

Hydrazine is an extremely toxic compound and contained high potential energy, considered as a rocket fuel. The hydrazine has not been found in microorganism synthesis due to its high toxicity.

This unique compound firstly was found in the Anammox bacteria. The Anammox bacteria have a special membrane “anammoxosome” to resist the toxicity of hydrazine. The synthesis pathway of ammonium and nitrite in Anammox was shown in Fig. 6. The electron acceptor nitrite is reduced to hydroxylamine, which reacted with ammonium as electron donor for forming hydrazine. The end product of Anammox was oxidation of hydrazine to produce nitrogen gas and provides four electrons to reduce nitrite for forming hydroxylamine. The oxidation and reduction enzymes involved in the reaction include hydrazine dydrolase (HH), hydrazine-oxidizing enzyme

Parameters Nitrification Anammox

Biomass yield 0.08 mol C (mol

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(HZO), and nitrite-reducing enzyme (NR)[27].

Fig. 6 Mechanism of Anammox [27].