The current perception of evolutionary relationships and the natural diversity of ammonia-oxidizing bacteria (AOB) is mainly based on comparative sequence analyses of their genes encoding the 16S rRNA and the active site polypeptide of the ammonia monooxygenase (AmoA). However, only partial 16S rRNA sequences are available for many AOB species and most AOB have not yet been analyzed on the amoA level. In this study, the 16S rDNA sequence data of 10 Nitrosomonas species and Nitrosococcus mobilis were completed. Furthermore, previously unavailable 16S rRNA sequences were determined for three Nitrosomonas sp. isolates and for the gamma-subclass proteobacterium Nitrosococcus halophilus. These data were used to revaluate the specificities of published oligonucleotide primers and probes for AOB. In addition, partial amoA sequences of 17 AOB, including the above-mentioned 15 AOB, were obtained. Comparative phylogenetic analyses suggested similar but not identical evolutionary relationships of AOB by using 16S rRNA and AmoA as marker molecules, respectively. The presented 16S rRNA and amoA and AmoA sequence data from all recognized AOB species significantly extend the currently used molecular classification schemes for AOB and now provide a more robust phylogenetic framework for molecular diversity inventories of AOB. For 16S rRNA-independent evaluation of AOB species-level diversity in environmental samples, amoA and AmoA sequence similarity threshold values were determined which can be used to tentatively identify novel species based on cloned amoA sequences. Subsequently, 122 amoA sequences were obtained from 11 nitrifying wastewater treatment plants. Phylogenetic analyses of the molecular isolates showed that in all but two plants only nitrosomonads could be detected. Although several of the obtained amoA sequences were only relatively distantly related to known AOB, none of these sequences unequivocally suggested the existence of previously unrecognized species in the wastewater treatment environments examined.Chemolithoautotrophic ammonia-oxidizing bacteria (AOB) play a central role in the natural cycling of nitrogen by aerobically transforming ammonia to nitrite. From an anthropocentric point of view, the activity of AOB is considered to be both detrimental and beneficial. AOB oxidize urea and ammonia fertilizers to nitrite and, in conjunction with nitrite oxidizers which subsequently convert nitrite to nitrate, thus contribute to fertilizer loss from agricultural soils by producing compounds which are easily washed out or used as electron acceptors for denitrification (42). The former process is also responsible for significant pollution of water supplies with nitrite and nitrate. Furthermore, AOB can produce greenhouse gases (8, 74) and corrode, because of the produced acid, stonework and concrete (46). On the other hand, AOB activity is encouraged in wastewater treatment plants to reduce the ammonia content of sewage before discharge into the receiving waters (49). Reduction of ammonia releases into aquatic en...

Chlamydiae are the major cause of preventable blindness and sexually transmitted disease. Genome analysis of a chlamydia-related symbiont of free-living amoebae revealed that it is twice as large as any of the pathogenic chlamydiae and had few signs of recent lateral gene acquisition. We showed that about 700 million years ago the last common ancestor of pathogenic and symbiotic chlamydiae was already adapted to intracellular survival in early eukaryotes and contained many virulence factors found in modern pathogenic chlamydiae, including a type III secretion system. Ancient chlamydiae appear to be the originators of mechanisms for the exploitation of eukaryotic cells.

The phylogenetic relationship of 12 ammonia-oxidizing isolates (eight nitrosospiras and four nitrosomonads), for which no gene sequence information was available previously, was investigated based on their genes encoding 16S rRNA and the active site subunit of ammonia monooxygenase (AmoA). Almost full-length 16S rRNA gene sequences were determined for the 12 isolates. In addition, 16S rRNA gene sequences of 15 ammonia-oxidizing bacteria (AOB) published previously were completed to allow for a more reliable phylogeny inference of members of this guild. Moreover, sequences of 453 bp fragments of the amoA gene were determined from 15 AOB, including the 12 isolates, and completed for 10 additional AOB. 16S rRNA gene and amoA-based analyses, including all available sequences of AOB pure cultures, were performed to determine the position of the newly retrieved sequences within the established phylogenetic framework. The resulting 16S rRNA gene and amoA tree topologies were similar but not identical and demonstrated a superior resolution of 16S rRNA versus amoA analysis. While 11 of the 12 isolates could be assigned to different phylogenetic groups recognized within the betaproteobacterial AOB, the estuarine isolate Nitrosomonas sp. Nm143 formed a separate lineage together with three other marine isolates whose 16S rRNA sequences have not been published but have been deposited in public databases. In addition, 17 environmentally retrieved 16S rRNA gene sequences not assigned previously and all originating exclusively from marine or estuarine sites clearly belong to this lineage. INTRODUCTIONChemolithoautotrophic ammonia-oxidizing bacteria (AOB) are capable of gaining energy via conversion of ammonia to nitrite and are thus of considerable importance in the global nitrogen cycle. Almost all aerobic environments in which organic matter is mineralized are possible habitats for AOB (Bock & Wagner, 2001). They have been detected in a variety of soil, marine, estuarine and freshwater systems and are crucial for the removal of nitrogen compounds in wastewater treatment plants (Painter, 1986), thus contributing to the impairment of anthropogenic damage to the environment. On the other hand, AOB activity causes deterioration of natural building stones (Bock & Sand, 1993) and enhances nitrogen fertilizer loss from arable soil (MacDonald, 1986). Due to their importance in natural and engineered systems, significant efforts have been made to characterize the diversity, distribution patterns and ecophysiology of AOB (for reviews see Koops & Pommerening-Röser, 2001; Koops et al., 2003).The first isolation of AOB was reported in 1890 (Frankland & Frankland, 1890;Winogradsky, 1890) and since then a considerable number of AOB isolates was obtained from various environments, leading to the description of 16 AOB species (reviewed by Koops et al., 2003). Comparative 16S rRNA gene sequence analyses of these species showed that 'Nitrosococcus halophilus' and Nitrosococcus oceani belong to the class 'Gammaproteobacteria', while the remaining...

The microbial community structure and activity dynamics of a phosphate-removing biofilm from a sequencing batch biofilm reactor were investigated with special focus on the nitrifying community. O 2 , NO 2 ؊ , and NO 3 ؊ profiles in the biofilm were measured with microsensors at various times during the nonaerated-aerated reactor cycle. In the aeration period, nitrification was oxygen limited and restricted to the first 200 m at the biofilm surface. Additionally, a delayed onset of nitrification after the start of the aeration was observed. Nitrate accumulating in the biofilm in this period was denitrified during the nonaeration period of the next reactor cycle. Fluorescence in situ hybridization (FISH) revealed three distinct ammonia-oxidizing populations, related to the Nitrosomonas europaea, Nitrosomonas oligotropha, and Nitrosomonas communis lineages. This was confirmed by analysis of the genes coding for 16S rRNA and for ammonia monooxygenase (amoA). Based upon these results, a new 16S rRNA-targeted oligonucleotide probe specific for the Nitrosomonas oligotropha lineage was designed. FISH analysis revealed that the first 100 m at the biofilm surface was dominated by members of the N. europaea and the N. oligotropha lineages, with a minor fraction related to N. communis. In deeper biofilm layers, exclusively members of the N. oligotropha lineage were found. This separation in space and a potential separation of activities in time are suggested as mechanisms that allow coexistence of the different ammonia-oxidizing populations. Nitrite-oxidizing bacteria belonged exclusively to the genus Nitrospira and could be assigned to a 16S rRNA sequence cluster also found in other sequencing batch systems.Modern biological treatment of wastewater involves not only C removal, but also elimination of the nutrients P and N (5,20). This requires the combined or sequential actions of various groups of microorganisms, such as heterotrophic bacteria, phosphate-accumulating organisms (PAO), and nitrifying and denitrifying bacteria. Consequently, purification plants and processes have become increasingly complex to satisfy the needs of the different microorganisms, usually in several reactor stages (5, 27). The integration of different functions in a single reactor would save reaction space and time and therefore is desirable from an economical point of view. However, difficulties often arise in establishing stable nitrification in such complex systems. Nitrifying bacteria (i.e., ammonia-oxidizing bacteria [AOB] and nitrite-oxidizing bacteria [NOB]) usually show low maximum growth rates, relatively low substrate affinities, and high sensitivity to toxic shocks or sudden pH changes (17,25,41). In the presence of organic matter, they can be easily outcompeted by heterotrophs for oxygen (56) and ammonia (19). Other problems to be solved are the inhibition of denitrification by the presence of oxygen (5) and the need for cyclic changes of oxic and anoxic (i.e., free of oxygen and nitrate) conditions for biological phosphate removal...

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Two biofilm reactors operated with hydraulic retention times of 0.8 and 5.0 h were used to study the links between population dynamics and reactor operation performance during a shift in process operation from pure nitrification to combined nitrification and organic carbon removal. The ammonium and the organic carbon loads were identical for both reactors. The composition and dynamics of the microbial consortia were quantified by fluorescence in situ hybridization (FISH) with rRNA-targeted oligonucleotide probes combined with confocal laser scanning microscopy, and digital image analysis. In contrast to past research, after addition of acetate as organic carbon nitrification performance decreased more drastically in the reactor with longer hydraulic retention time. FISH analysis showed that this effect was caused by the unexpected formation of a heterotrophic microorganism layer on top of the nitrifying biofilm that limited nitrifiers oxygen supply. Our results demonstrate that extension of the hydraulic retention time might be insufficient to improve combined nitrification and organic carbon removal in biofilm reactors. r

Biological wastewater treatment has been applied for more than a century to ameliorate anthropogenic damage to the environment. But only during the last decade the use of molecular tools allowed to accurately determine the composition, and dynamics of activated sludge and biofilm microbial communities. Novel, in many cases yet not cultured bacteria were identified to be responsible for filamentous bulking and foaming as well as phosphorus and nitrogen removal in these systems. Now, methods are developed to infer the in situ physiology of these bacteria. Here we provide an overview of what is currently known about the identity and physiology of some of the microbial key players in activated sludge and biofilm systems.

The nitrifying microbial diversity and population structure of a sequencing biofilm batch reactor receiving sewage with high ammonia and salt concentrations (SBBR 1) was analyzed by the full-cycle rRNA approach. The diversity of ammonia-oxidizers in this reactor was additionally investigated using comparative sequence analysis of a gene fragment of the ammonia monooxygenase (amoA), which represents a key enzyme of all ammonia-oxidizers. Despite the "extreme" conditions in the reactor, a surprisingly high diversity of ammonia- and nitrite-oxidizers was observed to occur within the biofilm. In addition, molecular evidence for the existence of novel ammonia-oxidizers is presented. Quantification of ammonia- and nitrite-oxidizers in the biofilm by Fluorescent In situ Hybridization (FISH) and digital image analysis revealed that ammonia-oxidizers occurred in higher cell numbers and occupied a considerably larger share of the total biovolume than nitrite-oxidizing bacteria. In addition, ammonia oxidation rates per cell were calculated for different WWTPs following the quantification of ammonia-oxidizers by competitive PCR of an amoA gene fragment. The morphology of nitrite-oxidizing, unculturable Nitrospira-like bacteria was studied using FISH, confocal laser scanning microscopy (CLSM) and three-dimensional visualization. Thereby, a complex network of microchannels and cavities was detected within microcolonies of Nitrospira-like bacteria. Microautoradiography combined with FISH was applied to investigate the ability of these organisms to use CO2 as carbon source and to take up other organic substrates under varying conditions. Implications of the obtained results for fundamental understanding of the microbial ecology of nitrifiers as well as for future improvement of nutrient removal in wastewater treatment plants (WWTPs) are discussed.