Introduction to the Anammox Bacteria Process
Anaerobic ammonium oxidation (Anammox) bacteria are autotrophic bacteria that, under anoxic conditions, use ammonia as the electron donor and nitrite as the electron acceptor to produce nitrogen gas (N2). All discovered Anammox bacteria belong to the family Anammoxaceae of the order Planctomycetales, comprising 6 genera: Candidatus Brocadia, Candidatus Kuenenia, Candidatus Anammoxoglobus, Candidatus Jettenia, Candidatus Anammoximicrobium moscowii, and Candidatus Scalindua. Among them, Candidatus Scalindua was found in the marine suboxic zone and is called marine anammox bacteria, while the other 5 genera were all found in wastewater treatment systems and are called freshwater anammox bacteria. Anammox bacteria are of great significance to the global nitrogen cycle and are also important bacteria in wastewater treatment. [2]
Van de Graaf et al. used 15N as a tracer to study the anammox metabolic pathway. Based on the hypothesis that the conversion of N2H4 to N2 provides equivalent electrons to the reduction of NO2- to NH2OH, they proposed two possible mechanisms. First, a membrane-enclosed enzyme complex converts ammonia and NH2OH into N2H4, which is then oxidized to nitrogen gas in the periplasm; the electrons produced are transferred internally to reduce NO2- to NH2OH in the cytoplasm (this enzyme complex is also responsible for N2H4 oxidation). Second, ammonia and NH2OH are converted into N2H4 in the cytoplasm by a membrane-enclosed enzyme complex,
N2H4 is converted into N2 in the periplasm, and the electrons produced are transmitted through the electron transport chain to the nitrite reductase in the cytoplasm, reducing NO2- to NH2OH.
Based on the view that the key enzyme of the anammox reaction is the hydrazine oxidoreductase (HZO) located in the anammoxosome, a biochemical model related to the anammoxosome membrane was proposed: NH4+ and hydroxylamine (NH2OH) are converted into hydrazine by hydrazine hydrolase (HH), and hydrazine is then oxidized by hydrazine oxidoreductase (HZO), which is similar to HAO (N. europaea). The oxidation of hydrazine occurs inside the anammoxosome, forming N2, 4 protons, and 4 electrons. These 4 electrons, together with 5 protons from the riboplasm, reduce nitrite to hydroxylamine through nitrite reductase (NIR). In this model, the anammox reaction establishes a proton gradient through proton consumption in the riboplasm and proton generation inside the anammoxosome. This creates an electrochemical proton gradient between the anammoxosome and the riboplasm. This gradient contains chemical potential energy (ΔpH) and electrical potential energy.
The chemical and electrical potential energies generate a proton motive force Δp that drives protons to move from inside the anammoxosome to outside. Under the catalysis of the anammoxosome-membrane-bound adenosine triphosphatase (ATPase), adenosine triphosphate (ATP) is synthesized. Protons passively migrate back into the riboplasm through the proton pore formed by ATPase; the anammoxosome-membrane-bound
ATPase is located in the spherical hydrophilic ATP synthesis region of the riboplasm and the hydrophobic proton-transport region of the anammoxosome membrane, and the synthesized ATP is released into the riboplasm.
(1) Candidatus “Brocadia anammoxidans”
Strous et al. studied the physiological parameters of anammox sludge (dominant bacterium B. anammoxidans) in an SBR reactor. More than 70% of the enriched culture was a dominant autotrophic bacterium. After treatment with a pH 7.4, 20 mmol/L K2HPO4/KH2PO4 buffer and 2.5% glutaraldehyde mixture, it showed irregular microbial characteristics under an electron microscope. A very pure cell suspension was obtained by an improved Percoll density gradient centrifugation method, with fewer than 1 heterotrophic bacterium per 200-800 bacteria. These purified anammox bacteria were highly active; DNA extracted from them was confirmed by PCR amplification and 16S rRNA analysis to show that B. anammoxidans is the functional microorganism of anammox, confirming that anammox bacteria are a new member of autotrophs in the Planctomycetales sequence, named Candidatus “B. anammoxida”.
(2) Candidatus “Kuenenia stuttgartiensis”
(3) Candidatus “Scalindua sorokinii”
... 13 mmol/L, active even at lower cell density. The maximum anammox activity (per unit protein) is 26.5 nmol/(mg·min), lower than that of B. anammoxidans. The pH range is 6.5-9.0, optimal at 8.0, and the optimal temperature is 37°C. The study found that when the temperature rises to 45°C, no anammox activity is observed, and when the temperature drops back to 37°C, the anammox activity does not recover; the activity at 11°C is about 24% of that at 37°C, indicating that it is suitable for a mesophilic, slightly alkaline environment.
Candidatus “K. stuttgartiensis” was found in the biofilm reactors of several wastewater treatment plants in Germany and Switzerland. Egli et al.'s research results show that K. stuttgartiensis acts similarly to B. anammoxidans, and electron microscopy shows its bacterial structure is also similar. However, it has higher tolerance to phosphate (20 mmol/L), and its tolerance to nitrite is
Recently, Candidatus “Scalindua sorokinii” was discovered in the Black Sea. It is further divided into two anammox species - Candidatus “Scalindua brodae” and Candidatus “Scalindua wagneri” - with relatively little research on their physiological characteristics. (4) Candidatus “Anammoxoglobus propionicus”
Candidatus “Anammoxoglobus propionicus” can metabolize propionate; its physiological characteristics are less studied.
During the anammox process, hydroxylamine and hydrazine act as intermediates of the metabolic process. Like other Planctomycetes, anammox bacteria also possess an intracellular membrane structure, within which the vesicle for anaerobic ammonia oxidation is called the anammoxosome, where the small, toxic hydrazine is generated. The membrane lipids of the anammoxosome have a special ladderane structure that prevents hydrazine leakage, thereby fully utilizing chemical energy and avoiding toxicity [3].
Anammox bacteria are diverse in morphology, appearing spherical, ovoid, etc., with a diameter of 0.8-1.1 μm. Anammox bacteria are Gram-negative. There is no capsule outside the cell. The cell wall surface has crater-like structures, and a few have pili. The cell is internally divided into 3 parts: the anammoxosome, the riboplasm
(riboplasm) and the paryphoplasm. The riboplasm contains ribosomes and the nucleoid, and most of the DNA is present here. The anammoxosome is a structure unique to anammox bacteria, accounting for 50%-80% of the cell volume, where the anammox reaction takes place. The anammoxosome is surrounded by a double membrane that deeply invaginates into the anammoxosome interior.
The cell wall of anammox bacteria is mainly composed of protein and contains no peptidoglycan. The cell membrane contains special ladderane membrane lipids, composed of multiple cyclobutane rings, shaped like a ladder. Among various anammox bacteria, the content of ladderane membrane lipids is basically similar. The hydrophobic ladderane membrane lipids combine with hydrophilic choline phosphate, ethanolamine phosphate, or glycerol phosphate to form phospholipids, constituting the skeleton of the cell membrane. The non-ladderane membrane lipids in the cell membrane are composed of straight-chain fatty acids, branched-chain fatty acids, monosaturated fatty acids, and triterpenoid compounds. It was once believed that ladderane membrane lipids existed only on the double membrane of the anammoxosome, with the function of limiting the diffusion of toxic intermediates. It is now believed that ladderane membrane lipids exist on all membrane structures of anammox bacteria (including the cytoplasmic membrane), and they combine with non-ladderane membrane lipids to ensure that the permeability of other membrane structures is better than that of the anammoxosome membrane.
Mulder et al. discovered anammox in an anaerobic fluidized bed. Later, Van de Graaf et al. and Bock et al. discovered the anammox process with nitrite as the electron acceptor. Zheng Ping et al. studied the kinetic characteristics of anammox bacterial mixed cultures. Fux Christian et al. conducted pilot-scale experimental research, first completing ammonia oxidation in a continuously stirred tank reactor, with 58% of NH4-N converted to NO2-; completing anammox in an SBR, with a nitrogen removal rate of 2.4 kg/(m³·d) and a nitrogen removal efficiency of 90%; Sliekers et al. found in an air-lift reactor a nitrogen removal rate of 8.9 kg/(m³·d), which is 20 times the nitrogen removal rate obtained in the laboratory. Dapena-Mora et al. found in their research that in an air-lift reactor the N loading rate was 2.0 g/(L·d), and the maximum specific anammox activity (MSAA) was 0.9 g/(g·d); in an SBR the N loading rate was 0.75 g/(L·d), MSAA was 0.4 g/(g·d), and the NO2- removal rate reached 99%.
Jetten et al. used the SHARON-ANAMMOX combined process to study sludge digestion effluent. The SHARON reactor had a total nitrogen loading of 0.8 kg/(m³·d), converting 53% of total nitrogen (39% NO2-, 14% NO3-); using the SHARON reactor effluent as the influent to the anammox fluidized-bed reactor, in the anammox reactor limited by NO2- all NO2- was removed, and the NH4-N removal rate reached 83% in the test. Van Dongen et al. applied the SHARON-ANAMMOX combined process for long-term stable operation in a plant.
Dijkman and Strous described a new biological nitrogen removal process, CANON, in which under oxygen-limited conditions (< 0.5% air saturation) a mixed culture of aerobic and anammox bacteria was obtained; NH4+ is oxidized to nitrite by aerobic ammonia-oxidizing bacteria (Nitrosomonas and Nitrosospira), and then converted to nitrogen gas by anammox bacteria. This process relies on the synergistic action of 2 autotrophic microbial communities (Nitrosomonas aerobic bacteria and Planctomycete anammox bacteria). CANON was studied in 2 different reactors (SBR and chemostat), with a volumetric loading (N) of 0.1 kg/(m³·d) and a nitrogen removal efficiency of 92%. Sliekers et al. found that under oxygen-limited conditions and with suitable loading rates for both aerobic ammonia-oxidizing bacteria and anammox bacteria, the nitrogen removal loading rate in the SBR reached 0.3 kg/(m³·d), with NH4+ mainly converted to N2 (85%) and the rest to nitrate (15%). Sliekers et al. used an air-lift reactor with a nitrogen removal loading rate of 1.5 kg/(m³·d), 20 times the rate previously obtained in the laboratory. Hao et al. developed a mathematical model of mixed nitrification (ammonia oxidation + nitrite oxidation) and anammox in a biofilm reactor, evaluating the temperature and flow velocity of the CANON process.
Jetten et al. removed COD through sludge digestion methanogenesis, partially oxidized N to NO2-, and then denitrified using NH4+ as the electron donor, achieving methanogenesis and anammox. Zhang applied EGSB reactor technology, achieving a COD removal rate of 97%, a NO2- removal rate of 100%, and a volumetric loading of 6.56 g/(L·d) (COD) and 0.99/(L·d) (N), realizing the coupling of methanogenesis, denitrification, and anammox [4].
