Ladderane lipid distribution in four genera of anammox bacteria

Intact ladderane phospholipids and core lipids were studied in four species of anaerobic ammonium oxidizing (anammox) bacteria, each representing one of the four known genera. Each species of anammox bacteria contained C(18) and C(20) ladderane fatty acids with either 3 or 5 linearly condensed cyclobutane rings and a ladderane monoether containing a C(20) alkyl moiety with 3 cyclobutane rings. The presence of ladderane lipids in all four anammox species is consistent with their putative physiological role to provide a dense membrane around the anammoxosome, the postulated site of anammox catabolism. In contrast to the core lipids, large variations were observed in the distribution of ladderane phospholipids, i.e. different combinations of hydrophobic tail (ladderane, straight chain and methyl branched fatty acid) types attached to the glycerol backbone sn-1 position, in combination with different types of polar headgroup (phosphocholine, phosphoethanolamine or phosphoglycerol) attached to the sn-3 position. Intact ladderane lipids made up a high percentage of the lipid content in the cells of "Candidatus Kuenenia stuttgartiensis", suggesting that ladderane lipids are also present in membranes other than the anammoxosome. Finally, all four investigated species contained a C(27) hopanoid ketone and bacteriohopanetetrol, which, indicates that hopanoids are anaerobically synthesised by anammox bacteria.     

Lipid composition of activated sludge in a pilot plant for anaerobic ammonium oxidation

The lipid composition of the microbial community inhabiting activated sludge in a pilot reactor for the anaerobic oxidation of ammonium (anammox) at the Kur’yanovo Treatment Plant (Moscow) has been studied. The fatty acid composition is mostly based on common fatty acids C₁₄–C₁₈ (95%) with both normal and isomeric structures. The biomass of activated sludge was found to contain lipids with the so-called ladderane substances (ladder alcohols and fatty acids) that are common for anammox bacteria: C₂₀-[3]-lad-derane and C₂₀-[5]-ladderane alcohols and C₁₈- and C₂₀-[3]-ladderane and C₁₈- and C₂₀-[5]-ladderane acids. In addition, the native extract contained both simple and compound ethers of the above-mentioned substances with residues of phosphocholine, phosphoethanolamine, and phosphoglycerine. The spectra of the electron impact and tandem mass spectrometry of certain substances have been obtained and published for the first time.     

Biophysical properties of membrane lipids of anammox bacteria: I. Ladderane phospholipids form highly organized fluid membranes

Anammox bacteria that are capable of anaerobically oxidizing ammonium (anammox) with nitrite to nitrogen gas produce unique membrane phospholipids that comprise hydrocarbon chains with three or five linearly condensed cyclobutane rings. To gain insight into the biophysical properties of these ‘ladderane’ lipids, we have isolated a ladderane phosphatidylcholine and a mixed ladderane phosphatidylethanolamine/phosphatidylglycerol lipid fraction and reconstituted these lipids in different membrane environments. Langmuir monolayer experiments demonstrated that the purified ladderane phospholipids form fluid films with a relatively high lipid packing density. Fluid-like behavior was also observed for ladderane lipids in bilayer systems as monitored by cryo-electron microscopy on large unilamellar vesicles (LUVs) and epi-fluorescence microscopy on giant unilamellar vesicles (GUVs). Analysis of the LUVs by fluorescence depolarization revealed a relatively high acyl chain ordering in the hydrophobic region of the ladderane phospholipids. Micropipette aspiration experiments were applied to study the mechanical properties of ladderane containing lipid bilayers and showed a relatively high apparent area compressibility modulus for ladderane containing GUVs, thereby confirming the fluid and acyl chain ordered characteristics of these lipids. The biophysical findings in this study support the previous postulation that dense membranes in anammox cells protect these microbes against the highly toxic and volatile anammox metabolites.     

Structural identification of ladderane and other membrane lipids of planctomycetes capable of anaerobic ammonium oxidation (anammox)

The membrane lipid composition of planctomycetes capable of the anaerobic oxidation of ammonium (anammox), i.e. Candidatus'Brocadia anammoxidans' and Candidatus'Kuenenia stuttgartiensis', was shown to be composed mainly of so-called ladderane lipids. These lipids are comprised of three to five linearly concatenated cyclobutane moieties with cis ring junctions, which occurred as fatty acids, fatty alcohols, alkyl glycerol monoethers, dialkyl glycerol diethers and mixed glycerol ether/esters. The highly strained ladderane moieties were thermally unstable, which resulted in breakdown during their analysis with GC. This was shown by isolation of a thermal product of these ladderanes and subsequent analysis with two-dimensional NMR techniques. Comprehensive MS and relative retention time data for all the encountered ladderane membrane lipids is reported, allowing the identification of ladderanes in other bacterial cultures and in the environment. The occurrence of ladderane lipids seems to be limited to the specific phylogenetic clade within the Planctomycetales able to perform anammox. This was consistent with their proposed biochemical function, namely as predominant membrane lipids of the so-called anammoxosome, the specific organelle where anammox catabolism takes place in the cell.     

Investigation of Proposed Ladderane Biosynthetic Genes from Anammox Bacteria by Heterologous Expression in E. coli

Ladderanes are hydrocarbon chains with three or five linearly concatenated cyclobutane rings that are uniquely produced as membrane lipid components by anammox (anaerobic ammonia-oxidizing) bacteria. By virtue of their angle and torsional strain, ladderanes are unusually energetic compounds, and if produced biochemically by engineered microbes, could serve as renewable, high-energy-density jet fuel components. The biochemistry and genetics underlying the ladderane biosynthetic pathway are unknown, however, previous studies have identified a pool of 34 candidate genes from the anammox bacterium, Kuenenia stuttgartiensis, some or all of which may be involved with ladderane fatty acid biosynthesis. The goal of the present study was to establish a systematic means of testing the candidate genes from K. stuttgartiensis for involvement in ladderane biosynthesis through heterologous expression in E. coli under anaerobic conditions. This study describes an efficient means of assembly of synthesized, codon-optimized candidate ladderane biosynthesis genes in synthetic operons that allows for changes to regulatory element sequences, as well as modular assembly of multiple operons for simultaneous heterologous expression in E. coli (or potentially other microbial hosts). We also describe in vivo functional tests of putative anammox homologs of the phytoene desaturase CrtI, which plays an important role in the hypothesized ladderane pathway, and a method for soluble purification of one of these enzymes. This study is, to our knowledge, the first experimental effort focusing on the role of specific anammox genes in the production of ladderanes, and lays the foundation for future efforts toward determination of the ladderane biosynthetic pathway. Our substantial, but far from comprehensive, efforts at elucidating the ladderane biosynthetic pathway were not successful. We invite the scientific community to take advantage of the considerable synthetic biology resources and experimental results developed in this study to elucidate the biosynthetic pathway that produces unique and intriguing ladderane lipids.     

Enrichment and characterization of marine anammox bacteria associated with global nitrogen gas production

Microbiological investigation of anaerobic ammonium oxidizing (anammox) bacteria has until now been restricted to wastewater species. The present study describes the enrichment and characterization of two marine Scalindua species, the anammox genus that dominates almost all natural habitats investigated so far. The species were enriched from a marine sediment in the Gullmar Fjord (Sweden) using a medium based on Red Sea salt. Anammox cells comprised about 90% of the enrichment culture after 10 months. The enriched Scalindua bacteria displayed all typical features known for anammox bacteria, including turnover of hydrazine, the presence of ladderane lipids, and a compartmentalized cellular ultrastructure. The Scalindua species also showed a nitrate-dependent use of formate, acetate and propionate, and performed a formate-dependent reduction of nitrate, Fe(III) and Mn(IV). This versatile metabolism may be the basis for the global distribution and substantial contribution of the marine Scalindua anammox bacteria to the nitrogen loss from oxygen-limited marine ecosystems.     

Biophysical properties of membrane lipids of anammox bacteria: II. Impact of temperature and bacteriohopanoids

Anammox bacteria possess unique membranes that are mainly comprised of phospholipids with extraordinary “ladderane” hydrocarbon chains containing 3 to 5 linearly concatenated cyclobutane moieties that have been postulated to form relatively impermeable membranes. In a previous study, we demonstrated that purified ladderane phospholipids form fluid-like mono- and bilayers that are tightly packed and relatively rigid. Here we studied the impact of temperature and the presence of bacteriohopanoids on the lipid density and acyl chain ordering in anammox membranes using Langmuir monolayer and fluorescence depolarization experiments on total lipid extracts. We showed that anammox membrane lipids of representatives of Candidatus “Kuenenia stuttgartiensis”, Candidatus “Brocadia fulgida” and Candidatus “Scalindua” were closely packed and formed membranes with a relatively high acyl chain ordering at the temperatures at which the cells were grown. Our findings suggest that bacteriohopanoids might play a role in maintaining the membrane fluidity in anammox cells.     

Isolation and characterization of a prokaryotic cell organelle from the anammox bacterium Kuenenia stuttgartiensis

Anaerobic ammonium oxidizing (anammox) bacteria oxidize ammonium with nitrite to nitrogen gas in the absence of oxygen. These microorganisms form a significant sink for fixed nitrogen in the oceans and the anammox process is applied as a cost‐effective and environment‐friendly nitrogen removal system from wastewater. Anammox bacteria have a compartmentalized cell plan that consists of three separate compartments. Here we report the fractionation of the anammox bacterium Kuenenia stuttgartiensis in order to isolate and analyze the innermost cell compartment called the anammoxosome. The subcellular fractions were microscopically characterized and all membranes in the anammox cell were shown to contain ladderane lipids which are unique for anammox bacteria. Proteome analyses and activity assays with the isolated anammoxosomes showed that these organelles harbor the energy metabolism in anammox cells. Together the experimental data provide the first thorough characterization of a respiratory cell organelle from a bacterium and demonstrate the essential role of the anammoxosome in the production of a major portion of the nitrogen gas in our atmosphere.     

Candidatus 'Brocadia fulgida': an autofluorescent anaerobic ammonium oxidizing bacterium

Anaerobic ammonium oxidizing (anammox) bacteria are detected in many natural ecosystems and wastewater treatment plants worldwide. This study describes the enrichment of anammox bacteria in the presence of acetate. The results obtained extend the concept that the anammox bacteria can be enriched to high densities in the presence of substrates for heterotrophic growth. Batch experiments showed that among the tested biomass, the biomass from the Candidatus 'Brocadia fulgida' enrichment culture oxidizes acetate at the highest rate. Continuous cultivation experiments showed that in the presence of acetate, ammonium, nitrite and nitrate, Candidatus 'Brocadia fulgida' out-competed other anammox bacteria. The results indicated that Candidatus 'Brocadia fulgida' did not incorporate acetate directly into their biomass. Candidatus 'Brocadia fulgida' exhibited the common characteristics of anammox bacteria: the presence of an anammoxosome and ladderane lipids and the production of hydrazine in the presence of hydroxylamine. Interestingly, the biofilm aggregates of this species showed strong autofluorescence. It is the only known anammox species exhibiting this feature. The autofluorescent extracellular polymeric substance had two excitation (352 and 442 nm) and two emission (464 and 521 nm) maxima.     

The anammoxosome: an intracytoplasmic compartment in anammox bacteria

Anammox bacteria belong to the phylum Planctomycetes and perform anaerobic ammonium oxidation (anammox); they oxidize ammonium with nitrite as the electron acceptor to yield dinitrogen gas. The anammox reaction takes place inside the anammoxosome: an intracytoplasmic compartment bounded by a single ladderane lipid-containing membrane. The anammox bacteria, first found in a wastewater treatment plant in The Netherlands, have the potential to remove ammonium from wastewater without the addition of organic carbon. Very recently anammox bacteria were also discovered in the Black Sea where they are responsible for 30-50% of the nitrogen consumption. This review will introduce different forms of intracytoplasmic membrane systems found in prokaryotes and discuss the compartmentalization in anammox bacteria and its possible functional relation to catabolism and energy transduction.     

Carbon isotope-labelling experiments indicate that ladderane lipids of anammox bacteria are synthesized by a previously undescribed, novel pathway

Ladderane lipids are unusual membrane lipids of bacteria that anaerobically oxidize ammonium to dinitrogen gas (anammox). Ladderane lipids contain linearly concatenated cyclobutane rings for which the pathway of biosynthesis is currently unknown. To investigate the possible biosynthetic routes of these lipids, 2-¹³C-labelled acetate was added to a culture of the anammox bacterium Candidatus Brocadia fulgida. Labelling patterns obtained by high-field ¹³C nuclear magnetic resonance spectroscopy of isolated lipids indicated that C . Brocadia fulgida synthesizes C_16:0 and iso C_16:0 fatty acids according to the known pathway of type II fatty acid biosynthesis. The ¹³C-labelling pattern of the C₈ alkyl chain of the C₂₀ [3] ladderane monoether also indicated the use of this route. However, carbon atoms in the cyclobutane rings and the cyclohexane ring were nonspecifically labelled and did not correspond to known patterns of fatty acid synthesis. Taken together, our results indicate that it is unlikely that ladderane lipids are formed from the cyclization of polyunsaturated fatty acids as hypothesized previously and suggest an alternative, although as yet unknown, pathway of biosynthesis.     

厌氧氨氧化菌脱氮机理及其在污水处理中的应用

厌氧氨氧化细菌(anammox)可以将亚硝酸盐和氨氮转化为氮气从而缩短氨氮转化的过程,它已经成为新型生物污水脱氮技术研究的热点之一。当前,有关厌氧氨氧化菌特有的生理结构特点、种群分类及其功能酶等方面的研究取得了一定突破,为实现其工业应用奠定了良好的理论基础;同时分子生物学技术在厌氧氨氧化细菌种群分布、群落多样性及其共生关系等方面的应用也大大促进了污水生物脱氮技术的革新和进步。总结了厌氧氨氧化菌主要的生理生化特点、细胞结构特点、脱氮机理、污水处理体系中的应用以及分子生物学方法对污水处理体系中厌氧氨氧化菌种群分析的研究现状,并指出未来anammox细菌在生物特性及在污水脱氮处理实际应用的研究中的热点问题。生物特性方面的主要研究热点有：（1）anammox细菌除厌氧氨氧化作用外,其它新陈代谢途径有待探索;（2）anammox细菌在不同环境中分布的倾向性问题;（3）新型anammox细菌的确定。污水处理的实际应用方面的主要研究热点有：（1）anammox污泥的快速高效富集问题;（2）设计高特异性引物;（3）anammox细菌和其他微生物的共生关系。     

Bacterial lipid diversity

The glycerophospholipids phosphatidylethanolamine, phosphatidylglycerol (PG), and cardiolipin (CL) are major structural components of bacterial membranes. In some bacteria, phosphatidylcholine or phosphatidylinositol and its derivatives form part of the membrane. PG or CL can be modified with the amino acid residues lysine, alanine, or arginine. Diacylglycerol is the lipid anchor from which syntheses of phosphorus-free glycerolipids, such as glycolipids, sulfolipids, or homoserine-derived lipids initiate. Many membrane lipids are subject to turnover and some of them are recycled. Other lipids associated with the membrane include isoprenoids and their derivatives such as hopanoids. Ornithine-containing lipids are widespread in Bacteria but absent in Archaea and Eukarya. Some lipids are probably associated exclusively with the outer membrane of many bacteria, i.e. lipopolysaccharides, sphingolipids, or sulfonolipids. For certain specialized membrane functions, specific lipid structures might be required. Upon cyst formation in Azotobacter vinelandii, phenolic lipids are accumulated in the membrane. Anammox bacteria contain ladderane lipids in the membrane surrounding the anammoxosome organelle, presumably to impede the passage of highly toxic compounds generated during the anammox reaction. Considering that present knowledge on bacterial lipids was obtained from only a few bacterial species, we are probably only starting to unravel the full scale of lipid diversity in bacteria. This article is part of a Special Issue entitled: Bacterial Lipids edited by Russell E. Bishop.     

16S rRNA gene and lipid biomarker evidence for anaerobic ammonium-oxidizing bacteria (anammox) in California and Nevada hot springs

Anammox, the oxidation of ammonium with nitrite to dinitrogen gas under anoxic conditions, is an important process in mesophilic environments such as wastewaters, oceans and freshwater systems, but little is known of this process at elevated temperatures. In this study, we investigated anammox in microbial mats and sediments obtained from several hot springs in California and Nevada, using geochemical and molecular microbiological methods. Anammox bacteria-specific ladderane core lipids with concentrations ranging between 0.3 and 52 ng g⁻¹ sediment were detected in five hot springs analyzed with temperatures up to 65 °C. In addition, 16S rRNA gene analysis showed the presence of genes phylogenetically related to the known anammox bacteria Candidatus Brocadia fulgida, Candidatus Brocadia anammoxidans and Candidatus Kuenenia stuttgartiensis (96.5–99.8% sequence identity) in three hot springs with temperatures up to 52 °C. Our data indicate that anammox bacteria may be able to thrive at thermophilic temperatures and thus may play a significant role in the nitrogen cycle of hot spring environments.     

Structural elucidation of zwitterionic sugar cores from glycosphingolipids by nanoelectrospray ionization-ion-trap mass spectrometry

The use of electrospray ionization (ESI)-ion-trap mass spectrometry (ITMS) for analysis of zwitterionic, glycolipid-derived sugar cores of glycosphingolipids is described. The capability of the method to perform multiple steps of fragmentation (MS(n)) allows structural characterization of these compounds. No derivatization of the released oligosaccharides is necessary when using nano-ESI with sample solution flow rates of about 30 nL/min. Investigations of positive as well as negative ions in fragmentation experiments up to MS(4) permit determination of the sequence of sugar units, their linkage positions, and the exact location of the substituents phosphocholine and phosphoethanolamine. In the case of phosphocholine, chemical cleavage of this substituent was necessary to obtain all the linkage information. Approximately 150-250 ng of sample was needed for each analysis.     

New anaerobic, ammonium-oxidizing community enriched from peat soil

Anaerobic ammonium-oxidizing (anammox) bacteria have been recognized as an important sink for fixed nitrogen and are detected in many natural environments. However, their presence in terrestrial ecosystems has long been overlooked, and their contribution to the nitrogen cycling in natural and agricultural soils is currently unknown. Here we describe the enrichment and characterization of anammox bacteria from a nitrogen-loaded peat soil. After 8 months of incubation with the natural surface water of the sampling site and increasing ammonium and nitrite concentrations, anammox cells constituted 40 to 50% of the enrichment culture. The two dominant anammox phylotypes were affiliated with "Candidatus Jettenia asiatica" and "Candidatus Brocadia fulgida." The enrichment culture converted NH(4)(+) and NO(2)(-) to N(2) with the previously reported stoichiometry (1:1.27) and had a maximum specific anaerobic ammonium oxidation rate of 0.94 mmol NH(4)(+)·g (dry weight)(-1)·h(-1) at pH 7.1 and 32°C. The diagnostic anammox-specific lipids were detected at a concentration of 650 ng·g (dry weight)(-1), and pentyl-[3]-ladderane was the most abundant ladderane lipid.     

Impact of Temperature on Ladderane Lipid Distribution in Anammox Bacteria

Anaerobic ammonium-oxidizing (anammox) bacteria have the unique ability to synthesize fatty acids containing linearly concatenated cyclobutane rings, termed “ladderane lipids.” In this study we investigated the effect of temperature on the ladderane lipid composition and distribution in anammox enrichment cultures, marine particulate organic matter, and surface sediments. Under controlled laboratory conditions we observed an increase in the amount of C₂₀ [5]-ladderane fatty acids compared with the amount of C₁₈ [5]-ladderane fatty acids with increasing temperature and also an increase in the amount of C₁₈ [5]-ladderane fatty acids compared with the amount of C₂₀ [5]-ladderane fatty acids with decreasing temperature. Combining these data with results from the natural environment showed a significant (R² = 0.85, P = <0.0001, n = 121) positive sigmoidal relationship between the amounts of C₁₈ and C₂₀ [5]-ladderane fatty acids and the in situ temperature; i.e., there is an increase in the relative abundance of C₁₈ [5]-ladderane fatty acids at lower temperatures and vice versa, particularly at temperatures between 12°C and 20°C. Novel shorter (C₁₆) and longer (C₂₂ to C₂₄) ladderane fatty acids were also identified, but their relative amounts were small and did not change with temperature. The adaptation of ladderane fatty acid chain length to temperature changes is similar to the regulation of common fatty acid composition in other bacteria and may be the result of maintaining constant membrane fluidity under different temperature regimens (homeoviscous adaptation). Our results can potentially be used to discriminate between the origins of ladderane lipids in marine sediments, i.e., to determine if ladderanes are produced in situ in relatively cold surface sediments or if they are fossil remnants originating from the warmer upper water column.Anaerobic ammonium-oxidizing (anammox) bacteria possess the unique ability to oxidize NH₄⁺ with NO₂⁻ to N₂ under anoxic conditions (42). Since the discovery of the anammox process in a wastewater treatment plant in the Netherlands (21), studies have indicated that anammox bacteria are omnipresent in low-oxygen environments around the world. Anammox therefore forms an important link in both the oceanic (4, 7, 17, 18, 31) and freshwater (14, 33) nitrogen cycles. Unlike other Planctomycetes, anammox bacteria contain a unique “organelle” called the anammoxosome (19, 37, 44-46). The membrane of this compartment contains unusual “ladderane” lipids (37). The core ladderane lipids consist of C₁₈ and C₂₀ fatty acids containing either 3 or 5 linearly concatenated cyclobutane rings, which are ester bound to a glycerol backbone or ether bound as alkyl chains (35). In addition, the intact polar lipids containing the core lipid structures may have different types of polar head groups, including phosphatidylcholine (PC), phosphatidylethanolamine (PE), or phosphatidylglycerol (PG) (1, 22). In silico density simulation modeling experiments with a ladderane lipid-containing membrane (glycerol-bound mixed ether-ester containing both ladderane moieties) have indicated that ladderane lipids could provide a denser cell membrane than conventional membrane lipids (37). Since the anammoxosome appears to be impenetrable to fluorophores, the ladderane membrane could function in cell energy conservation (37, 44).Experimental evidence has shown that anammox bacteria isolated from wastewater treatment reactors grow over a wide range of temperatures (20 to 43°C) and have an optimum temperature of about 35°C (39). In the natural environment the anammox process has been reported to occur at temperatures as low as −2.5°C in sea ice (5, 26) and as high as 70°C in hot springs and hydrothermal vent areas (3, 12). Furthermore, “Candidatus Scalindua spp.” has been successfully enriched from marine sediment (Gullmarsfjord, Sweden) in sequencing batch reactors at temperatures of 15 and 20°C (43). In other bacteria containing common fatty acids temperature adaptation can be achieved by (among other things) modifying the composition of the membrane bilayers to deal with alterations in membrane viscosity due to changes in temperature. This process has been well documented and is termed “homeoviscous adaptation”; i.e., the fatty acid composition is changed to maintain membrane fluidity (23, 27, 34, 40). Currently, it is not known how anammox bacteria, with their highly unusual ladderane lipids, react to temperature. To investigate this, we analyzed the ladderane lipid composition of anammox bacteria grown at different temperatures in sequencing batch reactors and in samples from different natural environments covering a wide range of temperatures.     

Dynamics in an unusual acyl carrier protein from a ladderane lipid-synthesizing organism

Anaerobic ammonium-oxidizing (anammox) bacteria express a distinct acyl carrier protein implicated in the biosynthesis of the highly unusual “ladderane” lipids these organisms produce. This “anammox-specific” ACP, or amxACP, shows several unique features such as a conserved FF motif and an unusual sequence in the functionally important helix III. Investigation of the protein's structure and dynamics, both in the crystal by ensemble refinement and by MD simulations, reveals that helix III adopts a rare six-residue-long 3₁₀-helical conformation that confers a large degree of conformational and positional variability on this part of the protein. This way of introducing structural flexibility by using the inherent properties of 3₁₀-helices appears unique among ACPs. Moreover, the structure suggests a role for the FF motif in shielding the thioester linkage between the protein's prosthetic group and its acyl cargo from hydrolysis.     

Structural analysis of the core oligosaccharide from Pasteurella multocida strain X73

The structure of the core oligosaccharide region of the lipopolysaccharide from the Pasteurella multocida strain X73 was elucidated. The lipopolysaccharide was subjected to a variety of degradative procedures. The structure of the purified oligosaccharide was established by monosaccharide and methylation analyses, NMR spectroscopy and mass spectrometry. The following structure illustrates a similar structure to the recently identified oligosaccharide from another P. multocida strain VP161, but with additional symmetrical substitution of the terminal galactose residues with phosphoethanolamine moieties, where based on the NMR data all sugars were found in pyranose ring forms and Kdo is 3-deoxy-alpha-D-manno-2-oct-2-ulosonic acid, l,D-alpha-Hep is l-glycero-D-manno-heptose, PEtn is phosphoethanolamine and PCho is phosphocholine.     

Biochemistry and molecular biology of anammox bacteria

Anaerobic ammonium-oxidizing (anammox) bacteria are one of the latest additions to the biogeochemical nitrogen cycle. These bacteria derive their energy for growth from the conversion of ammonium and nitrite into dinitrogen gas in the complete absence of oxygen. These slowly growing microorganisms belong to the order Brocadiales and are affiliated to the Planctomycetes. Anammox bacteria are characterized by a compartmentalized cell architecture featuring a central cell compartment, the “anammoxosome”. Thus far unique “ladderane” lipid molecules have been identified as part of their membrane systems surrounding the different cellular compartments. Nitrogen formation seems to involve the intermediary formation of hydrazine, a very reactive and toxic compound. The genome of the anammox bacterium Kuenenia stuttgartiensis was assembled from a complex microbial community grown in a sequencing batch reactor (74% enriched in this bacterium) using a metagenomics approach. The assembled genome allowed the in silico reconstruction of the anammox metabolism and identification of genes most likely involved in the process. The present anammox pathway is the only one consistent with the available experimental data, thermodynamically and biochemically feasible, and consistent with Ockham’s razor: it invokes minimum biochemical novelty and requires the fewest number of biochemical reactions. The worldwide presence of anammox bacteria has now been established in many oxygen-limited marine and freshwater systems, including oceans, seas, estuaries, marshes, rivers and large lakes. In the marine environment over 50% of the N₂ gas released may be produced by anammox bacteria. Application of the anammox process offers an attractive alternative to current wastewater treatment systems for the removal of ammonia-nitrogen. Currently, at least five full scale reactor systems are operational.