What Are Anammox Bacteria? A Detailed Explanation of Wastewater-Treatment Processes
Anammox (anaerobic ammonium oxidation) bacteria are autotrophic bacteria that, under anoxic conditions, use ammonia as the electron donor and nitrite as the electron acceptor to produce . All known 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 is found in the marine suboxic zone and is called the marine anammox bacterium, while the other 5 genera are found in wastewater treatment systems and are called freshwater anammox bacteria. Anammox bacteria are important for the global nitrogen cycle and are also important bacteria in wastewater treatment. [2]
Van de Graaf et al. used N as a tracer element to study the anammox metabolic pathway. Based on the assumption that the conversion of N2H4 to N2 provides equal electrons to the reaction reducing N02 to NH2OH, they proposed two possible mechanisms. First, a membrane-bound enzyme complex converts ammonia and NH2OH into N2H4, which is oxidized to nitrogen in the periplasm, and the electrons produced are transferred internally to reduce N02 to NH2OH in the cytoplasm via an enzyme complex (which also handles N2H4 oxidation). Second, ammonia and NH2OH are converted to N2H4 in the cytoplasm by a membrane-bound enzyme complex,
N2H4 is converted to N2 in the periplasm, and the produced electrons are passed via the electron transport chain to the nitrite reductase in the cytoplasm to reduce N02 to NH2OH.
Based on the view that the key enzyme of the anammox reaction, hydrazine oxidoreductase (HZO), is located in the anammoxosome, a biochemical model related to the anammoxosome membrane was proposed: NH4 and hydroxylamine (NH2OH) are converted to hydrazine by hydrazine hydrolase (HH), and hydrazine is oxidized by HZO, which is similar to HAO (N. europaea). Hydrazine oxidation 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 via nitrite reductase (NIR). In this model, anammox 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 (delta pH) and electrical potential.
The chemical and electrical potentials generate a proton motive force delta p that drives protons from inside the anammoxosome to outside. Under the catalysis of the anammoxosome-membrane-bound ATP synthase (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 lies in the spherical hydrophilic ATP-synthesis region of the riboplasm and the hydrophobic proton-translocation region of the anammoxosome membrane; 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 bacteria were a dominant autotroph. After treatment with a pH 7.4 buffer of 20 mmol/L K2HPO4/KH2PO4 and 2.5% glutaraldehyde, they showed irregular microbial characteristics under the electron microscope. Using an improved Percoll density-gradient centrifugation, very pure cell suspensions were obtained, with fewer than 1 foreign bacterium per 200-800 bacteria. These purified anammox bacteria were highly active; DNA extracted from them, analyzed by PCR amplification and 16S rRNA, proved B. anammoxidans to be the functional microorganism of anammox and confirmed anammox bacteria as a new member of autotrophs in the Planctomycetales sequence, named Candidatus 'B. anammoxida'.
(2)Candidatus “Kuenenia stuttgartiensis”
Candidatus 'K. stuttgartiensis' was found in biofilm reactors of several wastewater treatment plants in Germany and Switzerland. Egli et al. showed that K. stuttgartiensis acts similarly to B. anammoxidans and its bacterial structure is also similar under the electron microscope. But it has higher phosphate tolerance (20 mmol/L) and nitrite tolerance of
13 mmol/L, and is active at lower cell density. Its 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, optimum 8.0, and optimum temperature 37 C. It was found that at 45 C no anammox activity is observed, and when the temperature falls back to 37 C the activity does not recover; at 11 C the activity is about 24% of that at 37 C, showing it prefers a mesophilic, slightly alkaline environment.
(3)Candidatus“Scalindu sorokinii”
The recently discovered Candidatus 'Scalindua sorokinii' in the Black Sea. It is further divided into two anammox species - Candidatus 'Scalindua brodae' and Candidatus 'Scalindua wagneri' - whose physiological characteristics are little studied. (4) Candidatus 'Anammoxoglobus propionicus'
Candidatus 'Anammoxoglobus propionicus' can metabolize propionate; its physiological characteristics are little studied.
During anammox, hydroxylamine and hydrazine are intermediates of the metabolic process. Like other Planctomycetes, anammox bacteria also have an intracellular membrane structure; the vesicle where anaerobic ammonia oxidation occurs is called the anammoxosome, where the small, toxic hydrazine is generated. The anammoxosome membrane lipid has a special ladderane structure that prevents hydrazine leakage, thus making full use of chemical energy and avoiding toxicity [3].
Anammox bacteria are morphologically diverse, spherical, ovoid, etc., 0.8-1.1 micrometer in diameter. They 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 divided into 3 parts: the anammoxosome, the riboplasm
(riboplasm) and the paryphoplasm. The riboplasm contains ribosomes and the nucleoid, and most DNA is here. The anammoxosome is a structure unique to anammox bacteria, accounting for 50%-80% of cell volume, where the anammox reaction takes place. The anammoxosome is surrounded by a double membrane that dips deeply into the anammoxosome.
Jetten et al. studied sludge digestion effluent using the SHARON-ANAMMOX combined process. The SHARON reactor had a total nitrogen load of 0.8 kg/(m·d), converting 53% of total nitrogen (39% NO2, 14% NO3); using the SHARON effluent as influent to an anammox fluidized-bed reactor, NO2 was completely removed in the N02-limited anammox reactor, and NH4-N removal reached 83% in the test. Van Dongen et al. applied the SHARON-ANAMMOX combined process for long-term stable operation in a plant.
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 electron acceptor. Zheng Ping et al. studied the kinetic characteristics of anammox mixed cultures [14]. Fux Christian et al. conducted pilot-scale tests: ammonia oxidation was first completed in a continuous stirred tank reactor, with 58% of NH4-N converted to NO2; anammox was completed in an SBR, with a nitrogen removal rate of 2.4 kg/(m·d) and nitrogen removal of 90%; Sliekers et al. found a nitrogen removal rate of 8.9 kg/(m·d) in an airlift reactor, 20 times the rate obtained in the laboratory. Dapena-Mora et al. found in an airlift reactor an N load rate of 2.0 g/(L·d) and a maximum specific anammox activity (MSAA) of 0.9 g/(g·d); in an SBR the N load rate was 0.75 g/(L·d), MSAA 0.4 g/(g·d), and NO2 removal 99%.
The cell wall of anammox bacteria is mainly composed of protein and contains no peptidoglycan. The cell membrane contains special ladderane lipids, formed by multiple cyclobutane units, shaped like a ladder. The ladderane lipid content is roughly similar among anammox bacteria. The hydrophobic ladderane lipids combine with hydrophilic phosphorylcholine, phosphoethanolamine or glycerophosphate to form phospholipids that make up the membrane skeleton. The non-ladderane membrane lipids consist of straight-chain fatty acids, branched fatty acids, monounsaturated fatty acids and triterpenes. It was once thought that ladderane lipids exist only on the double membrane of the anammoxosome, functioning to limit diffusion of toxic intermediates. It is now believed that ladderane lipids exist on all membrane structures of anammox bacteria (including the cytoplasmic membrane), combined with non-ladderane lipids to ensure that other membrane structures are more permeable than the anammoxosome membrane.
Dijkman and Strous described a new biological nitrogen removal process, CANON, which under oxygen-limited conditions (<0.5% air saturation) obtains a co-culture of aerobic and anaerobic ammonia-oxidizing bacteria: NH4 is oxidized to nitrite by aerobic ammonia-oxidizing bacteria (Nitrosomonas and Nitrosospira), then converted to nitrogen gas by anammox bacteria; this process relies on the synergy of two autotrophic microbial groups (the aerobic Nitrosomonas and the Planctomycete anammox bacteria). CANON was studied in two reactors (SBR and chemostat) with a volumetric N load of 0.1 kg/(m·d) and nitrogen removal of 92%. Sliekers et al. found that under oxygen-limited conditions with suitable load rates for both aerobic and anaerobic ammonia oxidizers, the SBR achieved an N removal load of 0.3 kg/(m·d), with NH4 mainly converted to N2 (85%) and the rest to nitrate (15%). Sliekers et al. used an airlift reactor with an N removal load of 1.5 kg/(m·d), 20 times the rate previously obtained in the laboratory. Hao et al. [18] developed a mathematical model of combined 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 by sludge digestion methanogenesis, partially oxidized N to NO2, then denitrified using NH4 as electron donor, achieving methanization and anammox. Zhang used EGSB reactor technology with COD removal of 97%, NO2 removal of 100%, and volumetric loads of 6.56 g/(L·d) (COD) and 0.99/(L·d) (N), achieving coupling of methanization, denitrification and anammox [4].
