Brocadia Anammoxidans Research Articles

Identification of anammox bacteria in a full-scale deammonification plant making use of anaerobic ammonia oxidation

The existence of anaerobic ammonia-oxidizing (anammox) bacteria was postulated in the late 1970s. Approximately 20 years later, these lithotrophic members of the nitrogen cycle were identified as deep-branching members of the planctomycetes. Recently, full-scale implementation of biological deammonification was successfully achieved in the DEMON ® reactor at the wastewater treatment plant in Strass, Austria. The sludge of this reactor contains red granules and brownish flocs that can be physically separated. The two fractions yielded different banding patterns in denaturing gradient gel electrophoresis of PCR products obtained with primer sets targeting the 16S rRNA genes of planctomycetes. Comparative analysis of partial sequences of almost full-length 16S rRNA gene clones obtained from the granules and flocs confirms the differences in the community composition of the two fractions. The sequences retrieved from the red granules were 93% similar to those of Candidatus Brocadia anammoxidans, a bacterium known to catalyze the anaerobic ammonia oxidation.

Development of high-rate anaerobic ammonium-oxidizing (anammox) biofilm reactors

To promptly establish anaerobic ammonium oxidation (anammox) reactors, appropriate seeding sludge with high abundance and activity of anammox bacteria was selected by quantifying 16S rRNA gene copy numbers of anammox bacteria by real-time quantitative PCR (RTQ-PCR) and batch culture experiments. The selected sludge was then inoculated into up-flow fixed-bed biofilm column reactors with nonwoven fabric sheets as biomass carrier and the reactor performances were monitored over 1 year. The anammox reaction was observed within 50 days and a total nitrogen removal rate of 26.0 kg-N m −3 day −1 was obtained after 247 days. To our knowledge, such a high rate has never been reported before. Hydraulic retention time (HRT) and influent NH 4 + to NO 2 - molar ratio could be important determinant factors for efficient nitrogen removal in this study. The higher nitrogen removal rate was obtained at the shorter HRT and higher influent NH 4 + / NO 2 - molar ratio. After anammox reactors were fully developed, the community structure, spatial organization and in situ activity of the anammox biofilms were analyzed by the combined use of a full-cycle of 16S rRNA approach and microelectrodes. In situ hybridization results revealed that the probe Amx820-hybridized anaerobic anammox bacteria were distributed throughout the biofilm (accounting for more than 70% of total bacteria). They were associated with Nitrosomonas-like aerobic ammonia-oxidizing bacteria (AAOB) in the surface biofilm. The anammox bacteria present in this study were distantly related to the Candidatus Brocadia anammoxidans with the sequence similarity of 95%. Microelectrode measurements showed that a high in situ anammox activity (i.e., simultaneous consumption of NH 4 + and NO 2 - ) of 4.45 g-N of ( NH 4 + + NO 2 - ) m −2 day −1 was detected in the upper 800 μm of the biofilm, which was consistent with the spatial distribution of anammox bacteria.

Close relationship of RNase P RNA in Gemmata and anammox planctomycete bacteria

The relationship of RNase P RNA from anammox bacteria 'Candidatus Brocadia anammoxidans' and 'Candidatus Kuenenia stuttgartiensis' with that from other Planctomycetes was investigated. Newly identified rnpB gene sequences were aligned against existing planctomycete RNase P RNA sequences and secondary structures deduced by a comparative approach. Deduced secondary structures were similar in both anammox bacteria and both possessed an insert within the P13 helix analogous to that present in all Gemmata isolates. Phylogenetic analysis also revealed a possible relationship between the RNase P RNA molecules of the two anammox organisms and the genus Gemmata.

Quantification of anaerobic ammonium-oxidizing bacteria in enrichment cultures by real-time PCR

The anaerobic ammonium-oxidizing (ANAMMOX) bacteria were enriched from a rotating disk reactor (RDR) biofilm in semi-batch cultures. Based on fluorescence in situ hybridization (FISH) analysis, this enrichment led to a relative population size of 36% ANAMMOX bacteria. Phylogenetic analysis revealed that all the detected clones were related to the previously reported ANAMMOX bacteria, Candidatus Brocadia anammoxidans (AF375994), with 92% sequence similarity. Furthermore, we successfully developed a real-time polymerase chain reaction (PCR) assay to quantify populations of ANAMMOX bacteria in the enrichment cultures. For this real-time PCR assay, PCR primer sets targeting 16S ribosomal RNA genes of ANAMMOX bacteria were designed and used. The quantification range of this assay was 6 orders of magnitude, from 8.9×10 1 to 8.9×10 6 copies per PCR, corresponding to the detection limit of 3.6×10 3 target copies mL −1. A significant correlation was found between the increase in copy numbers of 16S rRNA gene of ANAMMOX bacteria and the increase in nitrogen removal rates in the enrichment cultures. Quantifying ANAMMOX bacterial populations in the enrichment culture made it possible to estimate the doubling time of the enriched ANAMMOX bacteria to be 3.6 to 5.4 days. The real-time PCR assay gave comparable population sizes in the enrichment cultures with the FISH results. These results suggest that the real-time PCR assay developed in this study is useful and reliable for quantifying the populations of ANAMMOX bacteria in environmental and engineering samples.

Anammox enrichment from different conventional sludges

Three sets of sequencing batch reactor (SBR) were used for Anammox enrichment from conventional sludges including upflow anaerobic sludge blanket, activated sludge, and anaerobic digestion sludge. After four months of operation, the Anammox activity occurred in all reactors allowing continuous removal of ammonium and nitrite. The morphology of the cultivated Anammox sludge was observed using scanning electron microscope. The photographs showed that the obtained culture was mostly spherical in shape, presumably Anammox culture. There were also filamentous-like bacteria co-existing in the system. Fluorescene in situ hybridization (FISH) analysis using 16S rRNA targeting oligonucleotide probes PLA46 and Amx820 showed that the dominant population developed in all SBRs was hybridized with both PLA46 and Amx820 gene probes. It means that the cultivated biomass in all SBRs was classified in the group of Planctomycetales bacteria with respect to the anaerobic ammonium-oxidizing bacteria, Candidatus Brocadia anammoxidans and Candidatus Kuenenia stuttgartiensis. Numerous time sequences were tested in this experiment. The shortest workable reaction time was found in the range from 5 to 7 h. Good quiescence of sludge was obtained at 30 min of settle period followed by a discharge period of 15 min. A long-term performance showed a near perfect removal of nitrite based on the influent NO 2 - – N concentration of 50–70 mg l −1. The maximum ammonia removal efficiency was 80% with the influent NH 4 + – N concentration of 40–60 mg l −1. It is, therefore, concluded that Anammox cultivation from conventional sludges was highly possible under control environment within four months.

Enantioselective Synthesis of Pentacycloanammoxic Acid

A highly effective enantioselective synthesis of pentacycloanammoxic acid (1), an unusual naturally occurring fatty acid from Candidatus Brocadia anammoxidans, has been accomplished. The C20-structure of 1 was assembled with stereocontrol from four building blocks, cyclobutene, 2-cyclopentenone, the chiral silylcyclopentenone 6, and 7-bromoheptanoic acid. Both 1 and its enantiomer are now available in quantities that should facilitate future studies on the mode of biosynthesis which appears to be unprecedented.

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.

Total Synthesis of (.+‐.)‐Pentacycloanammoxic Acid.

The first total synthesis of (+/-)-pentacycloanammoxic acid/methyl ester (1), an unusual naturally occurring fatty acid from Candidatus Brocadia anammoxidans, has been accomplished by the sequence of reactions shown in Scheme 1. The C20-structure of 1 was assembled with stereocontrol from three building blocks: cyclooctatetraene, 2-cyclopentenone, and 7-bromoheptanoic acid. The synthesis of 1 confirms the proposed structure of 1. The mode of biosynthesis and absolute configuration have yet to be clarified.

Biomarkers for in situ detection of anaerobic ammonium-oxidizing (anammox) bacteria.

The existence of anaerobic ammonium oxidation (anammox) was hypothesized based on nutrient profiles and thermodynamic calculations (5, 31, 44). It was first discovered about 1 decade ago (25) in a pilot plant treating wastewater from a yeast-producing company in Delft, The Netherlands. The anammox reaction is the oxidation of ammonium under anoxic conditions with nitrite as the electron acceptor and dinitrogen gas as the product. Hydroxylamine and hydrazine were identified as important intermediates (51). Due to their very low growth rates (doubling time in enrichments is at best 11 days) the cultivation of the anammox bacteria proved to be tedious and required very efficient biomass retention (41, 43). A physical purification of anammox organisms from enrichment cultures was achieved with percoll density centrifugation (42). The purified cells performed the anammox reaction after activation by hydrazine. Based on phylogenetic analysis, the discovered anammox organism branched deep in the Planctomycetes phylum (Fig. 1A and B, [42]) and was named “Candidatus Brocadia anammoxidans” (19). FIG. 1. (A) 16S rRNA gene-based phylogenetic tree reflecting the relationship of “Ca. Scalindua,” “Ca. Brocadia,” and “Ca. Kuenenia” to other Planctomycetes and other reference organisms. Tree reconstruction was ... After the first discovery, nitrogen losses, which could only be explained by the anammox reaction, were reported in other wastewater treatment facilities including landfill leachate treatment plants in Germany, Switzerland, and England (11, 14, 15, 36), as well as in semitechnical wastewater treatment plants in Germany (34), Belgium (30), Japan (12), Australia (48), and the United States (10, 45). Molecular techniques showed the presence of organisms affiliated with the anammox branch within the Planctomycetes in all these wastewater treatment plants. Nutrient profiles and 15N tracer studies in suboxic marine and estuarine environments indicated that anammox is also a key player in the marine nitrogen cycle (8, 46, 49). In addition, 16S rRNA gene analysis, fluorescence in situ hybridization (FISH), the distribution of specific anammox membrane lipids, nutrient profiles, and tracer experiments with [15N]ammonia showed the link between the anammox reaction and the occurrence of the anammox bacterium “Candidatus Scalindua sorokinii” in the suboxic zone of the Black Sea (20). The anammox reaction has also been tested for implementation for full-scale removal of ammonia in wastewater treatment (13, 52, 53). The detection and identification of active anammox organisms in environmental samples combined with information on environmental conditions can facilitate the search for possible biomass sources to be used as an inoculum for laboratory, semitechnical, or full-scale anammox reactors. Additionally, such information could provide insights into the niche differentiation of anammox organisms. This review summarizes the recent advances made in the 16S rRNA gene-based techniques for the detection of anammox bacteria. A convenient PCR detection method for anammox organisms is presented in which anammox-specific FISH probes were used as primers. Furthermore, methods which link activity and the detection of anammox bacteria, such as the combination of FISH and microautoradiography (FISH-MAR) (22) as well as FISH targeting the intergenic spacer region (ISR) between the 16S and 23S rRNA are discussed and compared to conventional methods to detect anammox activity. Each of these approaches by itself only addresses limited aspects, such as abundance, activity, or physiology. Thus, a combination of rRNA-based and non-rRNA-based methods is necessary to allow a comprehensive study of anammox bacteria in their ecosystems.

Enrichment of Anammox from Activated Sludge and Its Application in the CANON Process

A microbial culture capable of actively oxidizing ammonium to dinitrogen gas in the absence of oxygen, using nitrite as the electron acceptor, was enriched from local activated sludge (Western Australia) in <14 weeks. The maximum anaerobic ammonium oxidation (i.e., anammox) activity achieved by the anaerobic culture was 0.26 mmol NH (4) (+) (g biomass)(-1) h(-1) (0.58 kg total-N m(-3) day(-1)). Qualitative FISH analysis (fluorescence in situ hybridization) confirmed the phylogenetic position of the enriched microorganism as belonging to the order Planctomycetales, in which all currently identified anammox strains fall. Preliminary FISH analysis suggests the anammox strain belongs to the same phylogenetic group as the Candidatus 'Brocadia anammoxidans' strain discovered in the Netherlands. However, there are quite a few differences in the target sites for the more specific probes of these organisms and it is therefore likely to represent a new species of anammox bacteria. A small amount of aerobic ammonium-oxidizing biomass was inoculated into the anammox reactor (10% v/v) to initiate completely autotrophic nitrogen removal over nitrite (the CANON process) in chemostat culture. The culture was always under oxygen limitation and no organic carbon was added. The CANON reactor was operated as an intermittently aerated system with 20 min aerobiosis and 30 min anaerobiosis, during which aerobic and anaerobic ammonium oxidation were performed in sequential fashion, respectively. Anammox was not inhibited by repeated intermittent exposure to oxygen, allowing sustained, completely autotrophic ammonium removal (0.08 kg N m(-3) day(-1)) for an extended period of time.

1994–2004: 10 years of research on the anaerobic oxidation of ammonium

The obligately anaerobic ammonium oxidation (anammox) reaction with nitrite as primary electron acceptor is catalysed by the planctomycete-like bacteria Brocadia anammoxidans, Kuenenia stuttgartiensis and Scalindua sorokinii. The anammox bacteria use a complex reaction mechanism involving hydrazine as an intermediate. They have a unique prokaryotic organelle, the anammoxosome, surrounded by ladderane lipids, which exclusively contains the hydrazine oxidoreductase as the major protein to combine nitrite and ammonia in a one-to-one fashion. In addition to the peculiar microbiology, anammox was shown to be very important in the oceanic nitrogen cycle, and proved to be a very good alternative for treatment of high-strength nitrogenous waste streams. With the assembly of the K. stuttgartiensis genome at Genoscope, Evry, France, the anammox reaction has entered the genomic and proteomic era, enabling the elucidation of many intriguing aspects of this fascinating microbial process.

Implementation of the Anammox Process for Improved Nitrogen Removal

Stringent standards for nitrogen discharge necessitate the implementation of new systems for the sustainable removal of ammonium from wastewater. One of such systems is based on the process of anaerobic ammonium oxidation (Anammox), which is a new powerful tool especially for strong nitrogenous wastewaters. In this study, the Anammox process performance was tested with synthetic wastewater in a completely stirred tank reactor (CSTR). The reactor was operated for 511 days and fed with increasing amounts of ammonium and nitrite. In this period, an increase of ammonium and nitrite utilization rates were observed as a result of the increase of nitrogen loads in the influent. After 272 days, about 60% of the biomass was removed from the reactor and the system was restarted. Throughout 511 days 90% of the ammonium and more than 99% of the nitrite were converted mainly to dinitrogen (N2) and nitrate. The microbial community in the reactor was characterized with Fluorescence in situ Hybridization (FISH). The study showed that the population in the reactor was dominated by the deep-branching planctomycete Candidatus “Brocadia anammoxidans” strain Dokhaven 2.

Total Synthesis of (±)-Pentacycloanammoxic Acid

The first total synthesis of (+/-)-pentacycloanammoxic acid/methyl ester (1), an unusual naturally occurring fatty acid from Candidatus Brocadia anammoxidans, has been accomplished by the sequence of reactions shown in Scheme 1. The C20-structure of 1 was assembled with stereocontrol from three building blocks: cyclooctatetraene, 2-cyclopentenone, and 7-bromoheptanoic acid. The synthesis of 1 confirms the proposed structure of 1. The mode of biosynthesis and absolute configuration have yet to be clarified.

Application, eco-physiology and biodiversity of anaerobic ammonium-oxidizing bacteria

The demand for new and sustainable systems for nitrogen removal has increased dramatically in the last decade. It is clear that the conventional systems cannot deal with the increasing nitrogen loads in a cost effective way. As an alternative, the implementation of the anammox (anaerobic ammonium oxidation) process in the treatment of wastewater with high ammonium concentrations has been started. The compact anammox reactors can sustain high nitrogen loads without any problems. The highest observed anammox capacity is 8.9 kg N removed m-3 reactor day-1. The first 75 m3 anammox reactor is operating in Rotterdam, the Netherlands, combined with the partial nitrification process Single reaction system for High Ammonium Removal Over Nitrite (SHARON). Partial nitrification and anammox can also be combined in one reactor systems like Completely Autotrophic Nitrogen removal Over Nitrite (CANON) or Oxygen Limited Ammonium removal via Nitrification Denitrification (OLAND) where aerobic ammonium-oxidizing bacteria (AOB) and anammox bacteria cooperate under oxygen-limitation. These systems remove about 1.5 kg N m-3 reactor day-1. In addition to ammonium, urea can also be converted in the CANON system after a two-week adaptation period. The ecophysiological properties of the anammox bacteria make them very well suited to convert ammonium and nitrite. The Ks values for ammonium and nitrite are below 5 μM. However, nitrite above 10 mM is detrimental for the anammox process, and oxygen reversibly inhibits the process at concentrations as low as 1 μM. Acetate and propionate can be used by the anammox bacteria to convert nitrite and nitrate, whereas methanol and ethanol severely inhibit the anammox reaction. The enzyme hydroxylamine/hydrazine oxidoreductase (HAO), one of the key enzymes, is located in the anammoxosome, which is a membrane bound organelle. The membranes of the anammox bacteria contain unique ladderane lipids and hopanoids. The bacteria responsible for the anammox reaction are related to the Planctomycetes. The first anammox bacteria were isolated via Percoll centrifugation and characterized as Candidatus “Brocadia anammoxidans”. Survey of different wastewater treatment plants using anammox specific 16S rRNA gene primers and anammox specific oligonucleotide probes has revealed the presence of at least three other anammox bacteria, which have been tentatively named Candidatus “Kuenenia stuttgartiensis”, Candidatus “Scalindua wagneri” and Candidatus “Scalindua brodae”. A close relative of the latter, Candidatus “Scalindua sorokinii” was found to be responsible for about 50% of the nitrogen conversion in the anoxic zone of the Black Sea, making the anammox bacteria an important player in the oceanic nitrogen cycle.

Stable carbon isotopic fractionations associated with inorganic carbon fixation by anaerobic ammonium-oxidizing bacteria.

Isotopic analyses of Candidatus "Brocadia anammoxidans," a chemolithoautotrophic bacterium that anaerobically oxidizes ammonium (anammox), show that it strongly fractionates against (13)C; i.e., lipids are depleted by up to 47 per thousand versus CO(2). Similar results were obtained for the anammox bacterium Candidatus "Scalindua sorokinii," which thrives in the anoxic water column of the Black Sea, suggesting that different anammox bacteria use identical carbon fixation pathways, which may be either the Calvin cycle or the acetyl coenzyme A pathway.

活性汚泥からのＡＮＡＭＭＯＸ微生物の集積培養

Anaerobic ammonium removal was observed in batch tests using a denitrifying sludge from an industrial wastewater treatment plant and an intermittent nitrifying-denitrifying sludge from a mixed piggery domestic wastewater treatment plant after 92 and 84 days, respectively. The simultaneous removals of ammonium and nitrite with nitrate production at ratios corresponding to that reported for the Anammox process were observed. Their specific ammonium conversion rates following one year of incubation after the first detection were 0.260 and 0.281 g N·g protein-d-1, respectively. Using 16S rDNA sequence analysis, one of the sludges was confirmed to have an organism with a 99.2% similarity to the first reported Anammox bacterium, a deeply branching planctomycete, Candidatus Brocadia anammoxidans. In addition, the other sludge contained an organism with a 98.9% similarity to the Anammox planctomycete, Candidatus Kuenenia stuttgartiensis.

Anaerobic ammonium oxidation by marine and freshwater planctomycete-like bacteria.

Recently, two fresh water species, " Candidatus Brocadia anammoxidans" and " Candidatus Kuenenia stuttgartiensis", and one marine species, " Candidatus Scalindua sorokinii", of planctomycete anammox bacteria have been identified. " Candidatus Scalindua sorokinii" was discovered in the Black Sea, and contributed substantially to the loss of fixed nitrogen. All three species contain a unique organelle--the anammoxosome--in their cytoplasm. The anammoxosome contains the hydrazine/hydroxylamine oxidoreductase enzyme, and is thus the site of anammox catabolism. The anammoxosome is surrounded by a very dense membrane composed almost exclusively of linearly concatenated cyclobutane-containing lipids. These so-called 'ladderanes' are connected to the glycerol moiety via both ester and ether bonds. In natural and man-made ecosystems, anammox bacteria can cooperate with aerobic ammonium-oxidising bacteria, which protect them from harmful oxygen, and provide the necessary nitrite. The cooperation of these two groups of ammonium-oxidising bacteria is the microbial basis for a sustainable one reactor system, CANON (completely autotrophic nitrogen-removal over nitrite) to remove ammonia from high strength wastewater.

Anaerobic ammonia oxidation in the presence of nitrogen oxides (NO(x)) by two different lithotrophs.

The anaerobic ammonia-oxidizing activity of the planctomycete Candidatus "Brocadia anammoxidans" was not inhibited by NO concentrations up to 600 ppm and NO2 concentrations up to 100 ppm. B. anammoxidans was able to convert (detoxify) NO, which might explain the high NO tolerance of this organism. In the presence of NO2, the specific ammonia oxidation activity of B. anammoxidans increased, and Nitrosomonas-like microorganisms recovered an NO2-dependent anaerobic ammonia oxidation activity. Addition of NO2 to a mixed population of B. anammoxidans and Nitrosomonas induced simultaneous specific anaerobic ammonia oxidation activities of up to 5.5 mmol of NH4+ g of protein(-1) h(-1) by B. anammoxidans and up to 1.5 mmol of NH4+ g of protein(-1) h(-1) by Nitrosomonas. The stoichiometry of the converted N compounds (NO2-/NH3 ratio) and the microbial community structure were strongly influenced by NO2. The combined activity of B. anammoxidans and Nitrosomonas-like ammonia oxidizers might be of relevance in natural environments and for technical applications.

The anammox case-a new experimental manifesto for microbiological eco-physiology.

The anaerobic ammonium oxidation process is a new process for ammonia removal from wastewater. It is also a new microbial physiology that was previously believed to be impossible. The identification of Candidatus Brocadia anammoxidans and its relatives as the responsible bacteria was only possible with the development of a new experimental approach. That approach is the focus of this paper. The approach is a modernisation of the Winogradsky/Beyerinck strategy of selective enrichment and is based on the introduction of the molecular toolbox and modern bioreactor engineering to microbial ecology. It consists of five steps: (1) postulation of an ecological niche based on thermodynamic considerations and macro-ecological field data; (2) engineering of this niche into a laboratory bioreactor for enrichment culture; (3) black-box physiological characterisation of the enrichment culture as a whole; (4) phylogenetic characterisation of the enriched community using molecular tools; (5) physical separation of the dominant members of the enrichment culture using gradient centrifugation and the identification of the species of interest in accordance with Koch's postulates; (6) verification of the in situ importance of these species in the actual ecosystems. The power of this approach is illustrated with a case study: the identification of the planctomycetes responsible for anaerobic ammonium oxidation. We argue that this was impossible using molecular ecology or conventional 'cultivation based techniques' alone. We suggest that the approach might also be used for the microbiological study of many interesting microbes such as anaerobic methane oxidisers.

16S-23S rDNA intergenic spacer and 23S rDNA of anaerobic ammonium-oxidizing bacteria: implications for phylogeny and in situ detection.

Recently, anaerobic ammonium-oxidizing bacteria (AAOB) were identified by comparative 16S rDNA sequence analysis as a novel, deep-branching lineage within the Planctomycetales. This lineage consists currently of only two, not yet culturable bacteria which have been provisionally described as Candidatus 'Brocadia anammoxidans' and Candidatus 'Kuenenia stuttgartiensis'. In this study, a large fragment of the rDNA operon, including the 16S rDNA, the intergenic spacer region (ISR) and approximately 2 000 bases of the 23S rDNA, was polymerase chain reaction (PCR) amplified, cloned and sequenced from both AAOB. The retrieved 16S rDNA sequences of both species contain an insertion at helix 9 with a previously overlooked pronounced secondary structure (new subhelices 9a and 9b). This insertion, which is absent in all other known prokaryotes, is detectable by fluorescence in situ hybridization (FISH) and thus present in the mature 16S rRNA. In contrast with the genera Pirellula, Planctomyces and Gemmata that possess unlinked 16S and 23S rRNA genes, both AAOB have the respective genes linked together by an ISR of approximately 450 bp in length. Phylogenetic analysis of the obtained 23S rRNA-genes confirmed the deep branching of the AAOB within the Planctomycetales and allowed the design of additional specific FISH probes. Remarkably, the ISR of the AAOB also could be successfully detected by FISH via simultaneous application of four monolabelled oligonucleotide probes. Quantitative FISH experiments with cells of Candidatus 'Brocadia anammoxidans' that were inhibited by exposure to oxygen for different time periods demonstrated that the concentration of transcribed ISR reflected the activity of the cells more accurately than the 16S or 23S rRNA concentration. Thus the developed ISR probes might become useful tools for in situ monitoring of the activity of AAOB in their natural environment.