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.     

Anaerobic Ammonium-Oxidizing Bacteria: Unique Microorganisms with Exceptional Properties

Summary: Anaerobic ammonium-oxidizing (anammox) bacteria defy many microbiological concepts and share numerous properties with both eukaryotes and archaea. Among their most intriguing characteristics are their compartmentalized cell plan and archaeon-like cell wall. Here we review our current knowledge about anammox cell biology. The anammox cell is divided into three separate compartments by bilayer membranes. The anammox cell consists of (from outside to inside) the cell wall, paryphoplasm, riboplasm, and anammoxosome. Not much is known about the composition or function of both the anammox cell wall and the paryphoplasm compartment. The cell wall is proposed to be proteinaceous and to lack both peptidoglycan and an outer membrane typical of Gram-negative bacteria. The function of the paryphoplasm is unknown, but it contains the cell division ring. The riboplasm resembles the standard cytoplasmic compartment of other bacteria; it contains ribosomes and the nucleoid. The anammoxosome occupies most of the cell volume and is a so-called “prokaryotic organelle” analogous to the eukaryotic mitochondrion. This is the site where the anammox reaction takes place, coupled over the curved anammoxosome membrane, possibly giving rise to a proton motive force and subsequent ATP synthesis. With these unique properties, anammox bacteria are food for thought concerning the early evolution of the domains Bacteria, Archaea, and Eukarya.     

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.     

Linking ultrastructure and function in four genera of anaerobic ammonium-oxidizing bacteria: cell plan, glycogen storage, and localization of cytochrome C proteins

Anaerobic ammonium oxidation (anammox) is an ecologically and industrially important process and is performed by a clade of deeply branching Planctomycetes. Anammox bacteria possess an intracytoplasmic membrane-bounded organelle, the anammoxosome. In the present study, the ultrastructures of four different genera of anammox bacteria were compared with transmission electron microscopy and electron tomography. The four anammox genera shared a common cell plan and contained glycogen granules. Differences between the four genera included cell size (from 800 to 1,100 nm in diameter), presence or absence of cytoplasmic particles, and presence or absence of pilus-like appendages. Furthermore, cytochrome c proteins were detected exclusively inside the anammoxosome. This detection provides further support for the hypothesis that this organelle is the locus of anammox catabolism.     

Anammox bacteria in different compartments of recirculating aquaculture systems

Strict environmental restrictions force the aquaculture industry to guarantee optimal water quality for fish production in a sustainable manner. The implementation of anammox (anaerobic ammonium oxidation) in biofilters would result in the conversion of both ammonium and nitrite (both toxic to aquatic animals) into harmless dinitrogen gas. Both marine and freshwater aquaculture systems contain populations of anammox bacteria. These bacteria are also present in the faeces of freshwater and marine fish. Interestingly, a new planctomycete species appears to be present in these recirculation systems too. Further exploitation of anammox bacteria in different compartments of aquaculture systems can lead to a more environmentally friendly aquaculture practice.     

Anaerobic ammonium oxidation (anammox) in different natural ecosystems

Anammox (anaerobic ammonium oxidation), which is a reaction that oxidizes ammonium to dinitrogen gas using nitrite as the electron acceptor under anoxic conditions, was an important discovery in the nitrogen cycle. The reaction is mediated by a specialized group of planctomycete-like bacteria that were first discovered in man-made ecosystems. Subsequently, many studies have reported on the ubiquitous distribution of anammox bacteria in various natural habitats, including anoxic marine sediments and water columns, freshwater sediments and water columns, terrestrial ecosystems and some special ecosystems, such as petroleum reservoirs. Previous studies have estimated that the anammox process is responsible for 50% of the marine nitrogen loss. Recently, the anammox process was reported to account for 9-40% and 4-37% of the nitrogen loss in inland lakes and agricultural soils respectively. These findings indicate the great potential for the anammox process to occur in freshwater and terrestrial ecosystems. The distribution of different anammox bacteria and their contribution to nitrogen loss have been described in different natural habitats, demonstrating that the anammox process is strongly influenced by the local environmental conditions. The present mini-review summarizes the current knowledge of the ecological distribution of anammox bacteria, their contribution to nitrogen loss in various natural ecosystems and the effects of major influential factors on the anammox process.     

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。     

Cell biology of unique anammox bacteria that contain an energy conserving prokaryotic organelle

Anammox bacteria obtain their energy for growth from the anaerobic oxidation of ammonium with nitrite to dinitrogen gas. This property has made anammox bacteria very valuable for industry where they are applied for the removal of nitrogen compounds from industrial and domestic wastewaters. Anammox bacteria are also important in nature where they contribute significantly to oceanic nitrogen loss. Further, anammox bacteria have similarities to both Archaea and Eukarya, making them extremely interesting from a cell biological perspective. The anammox cell does not conform to the typical prokaryotic cell plan: single bilayer membranes divide the anammox cell into three distinct cellular compartments that possibly also have distinct cellular functions. The innermost and largest compartment, the anammoxosome, is the location of the energy metabolism. The middle compartment, the riboplasm, contains the nucleoid and ribosomes and thus has a genetic, information processing function. Finally, the outermost compartment, the paryphoplasm, has an as yet unknown function. In addition, anammox bacteria are proposed to have an atypical cell wall devoid of both peptidoglycan and a typical outer membrane. Here, I review the current knowledge on the cell biology of this enigmatic group of bacteria.     

Anaerobic Ammonium-Oxidizing (Anammox) Bacteria and Associated Activity in Fixed-Film Biofilters of a Marine Recirculating Aquaculture System

Microbial communities in the biological filter and waste sludge compartments of a marine recirculating aquaculture system were examined to determine the presence and activity of anaerobic ammonium-oxidizing (anammox) bacteria. Community DNA was extracted from aerobic and anaerobic fixed-film biofilters and the anaerobic sludge waste collection tank and was analyzed by amplifying 16S rRNA genes by PCR using anammox-selective and universal GC-clamped primers. Separation of amplified PCR products by denaturing gradient gel electrophoresis and sequencing of the different phylotypes revealed a diverse biofilter microbial community. While Planctomycetales were found in all three communities, the anaerobic denitrifying biofilters contained one clone that exhibited high levels of sequence similarity to known anammox bacteria. Fluorescence in situ hybridization studies using an anammox-specific probe confirmed the presence of anammox Planctomycetales in the microbial biofilm from the denitrifying biofilters, and anammox activity was observed in these biofilters, as detected by the ability to simultaneously consume ammonia and nitrite. To our knowledge, this is the first identification of anammox-related sequences in a marine recirculating aquaculture filtration system, and our findings provide a foundation for incorporating this important pathway for complete nitrogen removal in such systems.     

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.     

Anaerobic ammonium-oxidizing (anammox) bacteria and associated activity in fixed-film biofilters of a marine recirculating aquaculture system

Microbial communities in the biological filter and waste sludge compartments of a marine recirculating aquaculture system were examined to determine the presence and activity of anaerobic ammonium-oxidizing (anammox) bacteria. Community DNA was extracted from aerobic and anaerobic fixed-film biofilters and the anaerobic sludge waste collection tank and was analyzed by amplifying 16S rRNA genes by PCR using anammox-selective and universal GC-clamped primers. Separation of amplified PCR products by denaturing gradient gel electrophoresis and sequencing of the different phylotypes revealed a diverse biofilter microbial community. While Planctomycetales were found in all three communities, the anaerobic denitrifying biofilters contained one clone that exhibited high levels of sequence similarity to known anammox bacteria. Fluorescence in situ hybridization studies using an anammox-specific probe confirmed the presence of anammox Planctomycetales in the microbial biofilm from the denitrifying biofilters, and anammox activity was observed in these biofilters, as detected by the ability to simultaneously consume ammonia and nitrite. To our knowledge, this is the first identification of anammox-related sequences in a marine recirculating aquaculture filtration system, and our findings provide a foundation for incorporating this important pathway for complete nitrogen removal in such systems.     

Innovative treatment system for digester liquor using anammox process

This study demonstrated that partial nitritation using nitrifying activated sludge entrapped in a polyethylene glycol (PEG) gel carrier, as a pretreatment to anammox process, could be successfully applied to digester liquor of biogas plant at a nitrogen loading rate of 3.0 kg-N/m³/d. The nitritation process produced an effluent with a NO₂–N/NH₄–N ratio between 1.0 and 1.4, which was found to be suitable for the subsequent anammox process. A high SS concentration (2000–3000 mg/l) in the digester liquor did not affect partial nitritation treatment performances. Effluent from this partial nitritation reactor was successfully treated in the anammox reactor using anammox sludge entrapped in the PEG gel carrier with T-N removal rates of greater than 4.0 kg-N/m³/d. Influent BOD and SS contents did not inhibit anammox activity of the anammox gel carrier. The combination of partial nitritation and anammox reactors using PEG entrapped nitrifying and anammox bacteria was shown to be effective for the removal of high concentration ammonium in the digester liquor of a biogas plant.     

Anaerobic ammonium oxidation in a tropical freshwater system (Lake Tanganyika)

Here we provide the first direct evidence for the anammox process (anaerobic ammonium oxidation) in a lacustrine system, Lake Tanganyika, the second largest lake in the world. Incubations with (15)N labelled nitrate showed that anammox occurred in the suboxic water layer at 100-110 m water depth. Anammox rates up to 10 nM N(2) h(-1) are comparable to those reported for the marine water column. Up to approximately 13% of produced N(2) could be attributed to the anammox process whereas the remainder was related to denitrification. Typical lipid biomarkers characteristic of anammox bacteria were found in filtered water from the depths where anammox occurred, thus supporting the presence of anammox bacteria. Further evidence is provided by fluorescence in situ hybridization (FISH), revealing up to 13 000 anammox bacteria cells per ml or 1.4% of all DAPI (4'-6-Diamidino-2-phenylindole)-stained cells. Phylogenetic analyses of partial 16S rRNA genes indicated the presence of sequences most closely related to the known anammox bacterium Candidatus "Scalindua brodae" (95.7% similarity). Using the incubation results, a total loss of 0.2 Tg N(2) per year linked to anammox was estimated for the Northern basin of Lake Tanganyika.     

Application of anaerobic ammonium-oxidizing consortium to achieve completely autotrophic ammonium and sulfate removal

The simultaneous ammonium and sulfate removal was detected in an anammox reactor, consisted of ammonium oxidization with sulfate deoxidization, and subsequently traditional anammox process, in via of middle medium nitrite with solid sulfur and N2 as the terminal products. The pure anammox bacteria offered a great biotechnological potential for the completely autotrophic reaction indicated by batch tests. Denaturing gradient gel electrophoresis (DGGE) analysis further revealed that a new organism belonging to Planctomycetales was strongly enriched in the defined niche: the redox of ammonium and sulfate. The new species "Anammoxoglobussulfate" was so considered as holding a critical role in the ammonium oxidization with sulfate deoxidization to nitrite. Afterwards, the Planctomyces existing in the bacteria community performed the anammox process together to achieve the complete nitrogen and sulfate removal. The potential use of sulfate as electron acceptor for ammonium oxidizing widens the usage of anammox bacteria.     

厌氧氨氧化颗粒污泥聚集机制研究进展

厌氧氨氧化(anaerobic ammonium oxidation,anammox)工艺被认为是当前污水生物脱氮领域最经济的处理工艺,有利于实现污水处理厂的能源自给。厌氧氨氧化菌是该工艺的核心功能微生物。以厌氧氨氧化菌为主导微生物形成的厌氧氨氧化颗粒污泥具有沉速大、污泥持留能力强及对不利环境抵抗能力强等突出优势,是实现厌氧氨氧化工艺最有前景的污泥形态。本论文围绕厌氧氨氧化颗粒,介绍了厌氧氨氧化菌的特性、种类及代谢途径,综述了厌氧氨氧化颗粒污泥的形成假说及与厌氧氨氧化颗粒污泥聚集密切相关的胞外聚合物(extracellular polymeric substance,EPS)和群体感应研究现状,并对今后厌氧氨氧化颗粒的研究进行了展望,以期为后续厌氧氨氧化颗粒的研究及厌氧氨氧化颗粒工艺的优化提供参考。     

Enhanced anammox consortium activity for nitrogen removal: impacts of static magnetic field

Anaerobic ammonium oxidation (anammox) process has been becoming a promising technology for the removal of nitrogenous contaminants from wastewater. In short-term batch tests, we observed the anaerobic ammonium oxidizing activity of anammox consortium increased as the magnetic field varied in the range of 16.8-95.0mT. A maximum 50% increase was obtained at the value of 75.0mT. In order to study long-term effect of magnetic field on anammox consortium, an anammox reactor with magnetic field of 60.0mT was operated in laboratory-scale. The results demonstrated that a significant 30% increase in maximum nitrogen removal rate and an approximate 1/4 saving in cultivation time were achieved by using the magnetic system. Microbiological composition analysis showed that bacterial diversity in the reactor decreased under magnetic-exposed condition. Nevertheless, some strains belonging to Planctomycetales were highly enriched. These findings indicated that the magnetic field was useful and reliable for fast start-up of anammox process since it was proved as a simple and convenient approach to enhance anaerobic ammonium oxidizing activity.     

厌氧铵氧化过程中关键酶及相关分子标记在生态学研究中的应用进展

厌氧铵氧化是由微生物介导的氮素循环过程中的重要途径之一。近20年来,通过对厌氧铵氧化细菌生态学、基因组学和生理代谢特性的探索,人们对其微生物学机制已经有了较多的认识:厌氧铵氧化细菌通过亚硝酸盐还原酶将亚硝酸根离子还原为一氧化氮,进而与铵离子结合在联氨合成酶的作用下生成联氨,最后通过联氨氧化酶的催化产生终产物氮气。同时,对参与这些过程的关键酶及其功能基因的认识有助于选择新的分子标记,从而为研究厌氧铵氧化细菌的多样性和分子生态学特征提供新的工具,以弥补16S rRNA基因特异性相对较低且难以与生态功能关联等方面的不足。对目前已知的参与厌氧铵氧化过程的3种关键酶的研究历程和现状进行了评述,并总结了利用3种功能基因进行厌氧铵氧化细菌生态学研究的最新进展。     

Propionate Oxidation by and Methanol Inhibition of Anaerobic Ammonium-Oxidizing Bacteria

Anaerobic ammonium oxidation (anammox) is a recently discovered microbial pathway and a cost-effective way to remove ammonium from wastewater. Anammox bacteria have been described as obligate chemolithoautotrophs. However, many chemolithoautotrophs (i.e., nitrifiers) can use organic compounds as a supplementary carbon source. In this study, the effect of organic compounds on anammox bacteria was investigated. It was shown that alcohols inhibited anammox bacteria, while organic acids were converted by them. Methanol was the most potent inhibitor, leading to complete and irreversible loss of activity at concentrations as low as 0.5 mM. Of the organic acids acetate and propionate, propionate was consumed at a higher rate (0.8 nmol min⁻¹ mg of protein⁻¹) by Percoll-purified anammox cells. Glucose, formate, and alanine had no effect on the anammox process. It was shown that propionate was oxidized mainly to CO₂, with nitrate and/or nitrite as the electron acceptor. The anammox bacteria carried out propionate oxidation simultaneously with anaerobic ammonium oxidation. In an anammox enrichment culture fed with propionate for 150 days, the relative amounts of anammox cells and denitrifiers did not change significantly over time, indicating that anammox bacteria could compete successfully with heterotrophic denitrifiers for propionate. In conclusion, this study shows that anammox bacteria have a more versatile metabolism than previously assumed.