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Nitrite-oxidizing bacteria belonging to the genus Nitrospira mediate a key step in nitrification and play important roles in the biogeochemical nitrogen cycle and wastewater treatment. While these organisms have recently been shown to exhibit metabolic flexibility beyond their chemolithoautotrophic lifestyle, including the use of simple organic compounds to fuel their energy metabolism, the metabolic networks controlling their autotrophic and mixotrophic growth remain poorly understood. Here, we reconstructed a genome-scale metabolic model for Nitrospira moscoviensis (iNmo686) and used constraint-based analysis to evaluate the metabolic networks controlling autotrophic and formatotrophic growth on nitrite and formate, respectively. Subsequently, proteomic analysis and 13C-tracer experiments with bicarbonate and formate coupled to metabolomic analysis were performed to experimentally validate model predictions. Our findings support that N. moscoviensis uses the reductive tricarboxylic acid cycle for CO2 fixation. We also show that N. moscoviensis can indirectly use formate as a carbon source by oxidizing it first to CO2 followed by reassimilation, rather than direct incorporation via the reductive glycine pathway. Our study offers the first measurements of Nitrospira’s in vivo central carbon metabolism and provides a quantitative tool that can be used for understanding and predicting their metabolic processes.ImportanceNitrospira are globally abundant nitrifying bacteria in soil and aquatic ecosystems and wastewater treatment plants, where they control the oxidation of nitrite to nitrate. Despite their critical contribution to nitrogen cycling across diverse environments, detailed understanding of their metabolic network and prediction of their function under different environmental conditions remains a major challenge. Here, we provide the first constraint-based metabolic model of N. moscoviensis representing the ubiquitous Nitrospira lineage II and subsequently validate this model using proteomics and 13C-tracers combined with intracellular metabolomic analysis. The resulting genome-scale model will serve as a knowledge base of Nitrospira metabolism and lays the foundation for quantitative systems biology studies of these globally important nitrite- oxidizing bacteria.

Chemolithoautotrophic nitrite-oxidizing bacteria (NOB) of the genus Nitrospira contribute to nitrification in diverse natural environments and engineered systems. Nitrospira are thought to be well-adapted to substrate limitation owing to their high affinity for nitrite and capacity to use alternative energy sources. Here, we demonstrate that the canonical nitrite oxidizer Nitrospira moscoviensis oxidizes hydrogen (H2) below atmospheric levels using a high-affinity group 2a nickel-iron hydrogenase [Km(app) = 32 nM]. Atmospheric H2 oxidation occurred under both nitrite-replete and nitrite-deplete conditions, suggesting low-potential electrons derived from H2 oxidation promote nitrite-dependent growth and enable survival during nitrite limitation. Proteomic analyses confirmed the hydrogenase was abundant under both conditions and indicated extensive metabolic changes occur to reduce energy expenditure and growth under nitrite-deplete conditions. Respirometry analysis indicates the hydrogenase and nitrite oxidoreductase are bona fide components of the aerobic respiratory chain of N. moscoviensis, though they transfer electrons to distinct electron carriers in accord with the contrasting redox potentials of their substrates. Collectively, this study suggests atmospheric H2 oxidation enhances the growth and survival of NOB in amid variability of nitrite supply. These findings also extend the phenomenon of atmospheric H2 oxidation to a seventh phylum (Nitrospirota) and reveal unexpected new links between the global hydrogen and nitrogen cycles.

Global warming is predicted to alter the timing and magnitude of biogeochemical nitrogen cycling in paddy soils. However, little is known about its effect on active nitrifying populations. Here we investigated the responses of nitrification activity and active nitrifiers to elevated temperature in an acidic paddy soil. 13CO2-DNA-stable isotope probing (SIP), qPCR and high-throughput sequencing were used to determine active nitrifying phylotypes as well as difference in their abundance and community composition incubated at field temperature (15 °C) and elevated temperature (20 °C). Urea application led to significant production of nitrate and growth of ammonia-oxidizing bacteria (AOB) at both temperatures. Nitrification activity at elevated temperature was 148.3% and 18.5% higher than that of low temperature at day 28 and 56, respectively, accompanied by an increase in the extent of 13C-label incorporation by AOB. 13CO2-based SIP experiment indicated that both AOB and ammonia-oxidizing archaea (AOA) were involved in the nitrification activity and the active ammonia oxidizers changed from AOA to AOB with elevated temperature. Significant variation of AOA communities was observed under different temperatures. Dominant 13C-labeled nitrite-oxidizing bacteria (NOB) shifted from Nitrospira moscoviensis to Nitrospira japonica with higher temperature. Our findings emphasized that elevated temperature had pronounced effects on autotrophic nitrification which was mediated by altering relative abundance of active AOB and AOA, as well as the community composition of AOA and NOB. AOB were more adaptable than AOA with increasing abundance but no alteration of composition at elevated temperature.

The isolation of acid-adapted ammonia-oxidizing bacteria (AOB) has suggested the functional importance of AOB in acidic soils. However, there is, currently, no convincing evidence that links AOB activity to nitrification in acidic paddy soils. Here we demonstrated the incorporation of 13CO2 into the genomes of ammonia-oxidizing archaea (AOA), AOB and nitrite-oxidizing bacteria (NOB) following urea application by using stable isotope probing (SIP) in three acidic terrace paddy soils with altitudes of 200 m (E200), 600 m (E600) and 1100 m (E1100), respectively. Nitrification activity increased from E200 to E600 and then to E1100, accompanied with significant growth of AOB over the 56-day incubation, while the abundance of archaeal amoA gene declined significantly in all soils after incubation. DNA-SIP demonstrated that active AOB outnumbered AOA and were much more heavily labeled than AOA and NOB, implying their more significant contributions to nitrification in these soils. Phylogenetic analysis indicated that Nitrosospira cluster 3-like AOB predominantly catalyzed bacterial ammonia oxidation. 13C-labeled NOB was dominated by Nitrospira moscoviensis in E1100, while in E200 and E600, Nitrospira marina and Nitrospira japonica were as prevalent as Nitrospira moscoviensis, respectively. Canonical correlation analysis and the Mantel test indicated the importance of soil physiochemical properties (e.g., pH, available phosphorus (AP) and soil oxidation capacity (OXC)) in determining the composition of the active nitrifying populations. These results suggest a greater functional importance of AOB in ammonia oxidation in the tested acidic paddy soils and the existence of a broader ecological niche for AOB than previously considered.

Diese Bachelorarbeit beschäftigt sich mit der Charakterisierung der aeroben FormiatOxidation im Nitrit-oxidierenden Bakterium, Nitrospira moscoviensis. Nitrit-oxidierende Bakterien (NOB) gehören, genauso wie Ammoniak-oxidierende Bakterien (AOB) und Archaeen (AOA), sowie Comammox-Bakterien, zu den nitrifizierenden Mikroorganismen1,2 . Im Stickstoffkreislauf führen sie die Nitrifikation durch, die für die Erhaltung der Umwelt unerlässlich ist. Auch in der Trinkwasser- und Abwasserbehandlung sind sie von großem Nutzen, da sie unerwünschte Stickstoffverbindungen entfernen3-5. Durch ihr weit verbreitetes Vorkommen in der Umwelt und ihren Einfluss darauf, ist das Verständnis für ihre Stoffwechselprozesse ein großes Anliegen in der Umweltforschung. Ziel dieser Studie war die Ermittlung von kinetischen Parametern der Formiat-Oxidation in N. moscoviensis. Es wurden sowohl die Substrataffinität für Formiat (Km(app)), als auch die Maximalgeschwindigkeit der Formiat-Oxidation (Vmax) bestimmt. Darüber hinaus wurde ein Vergleich mit den kinetischen Parametern der Formiat-Oxidation im erst kürzlich entdeckten Comammox-Bakterium, Nitrospira inopinata, vorgenommen. Zur Beantwortung der Forschungsfragen wurden die Bakterien zunächst in einem Mineralsalzmedium kultiviert und nach Erreichen einer bestimmten Biomasse mittels Zentrifugation konzentriert. In einem Mikrorespirationssystem wurde anschließend der Sauerstoffverbrauch der Bakterien nach mehreren Formiat-Injektionen mit unterschiedlichen Konzentrationen gemessen. Bei den mikrorespiratorischen Messungen mit N. moscoviensis wurde ersichtlich, dass der Anstieg des Sauerstoffverbrauchs mit dem Anstieg der Formiat-Konzentrationen korreliert. Nachfolgend wurden die kinetischen Parameter von N. moscoviensis (Km(app) = 96.8 ± 4.9 μM and Vmax = 28.9 ± 7.1 μM h-1) und N. inopinata (Km(app) = 8777.6 ± 1106.6 μM and Vmax = 19.6 ± 1.2 μM h-1) miteinander verglichen. Aufgrund der Unterschiede zwischen den beiden Km(app)-Werten wurde somit verdeutlicht, dass zwar beide Nitrospira-Spezies in der Lage sind Formiat zu verwerten, N. moscoviensis jedoch eine viel stärkere Affinität zu Formiat aufweist als N. inopinata. This bachelor thesis deals with the characterization of the aerobic formate oxidation of the nitrite-oxidizing bacterium, Nitrospira moscoviensis. Nitrifying microorganisms, such as ammonia-oxidizing bacteria (AOB) and archaea (AOA), and nitrite-oxidizing bacteria (NOB) are important components of the environment, as they are involved in the nitrogen cycle and help to remove unwanted nitrogen compounds in drinking water and wastewater treatment plants1-5. Due to the widespread distribution of nitrifiers in the environment and the effects of their metabolic activities, understanding their metabolic pathways is of great importance for both natural and engineered systems. The aim of this study was to characterize the formate utilization by N. moscoviensis. For this purpose, the kinetic parameters, such as the substrate affinity for formate (Km(app)), as well as the maximal rate of formate oxidation (Vmax) were characterized. In addition, a comparison was made with the comammox bacteria Nitrospira inopinata. To answer the research questions, the bacteria were first cultured in a mineral salts medium specifically designed for NOB and concentrated after reaching a certain amount of biomass. Afterwards, the O2 consumption rate of the biomass was measured with an oxygen microsensor after injections with increasing formate concentrations. The results of the microrespiratory measurements with N. moscoviensis showed that an increase in O2 consumption rate correlated with the increase in formate concentrations. The kinetic parameters of N. moscoviensis (Km(app) = 96.8 ± 4.9 μM and Vmax = 28.9 ± 7.1 μM h-1) were then compared with the kinetic parameters of N. inopinata (Km(app) = 8777.6 ± 1106.6 μM and Vmax = 19.6 ± 1.2 μM h-1). Based on the enormous differences between the Km(app) values, it can be said that N. moscoviensis has a stronger affinity for formate than N. inopinata. In contrast to N. inopinata, a hyperbolic curve could be observed and the formate oxidation in N. moscoviensis follows a Michaelis-Menten model. Therefore, although both Nitrospira species are able to utilize formate, N. moscoviensis is a much stronger competitor for this alternative substrate.

Root-associated microbial communities play important roles in driving the microbial transformation of soil carbon and nitrogen cycling, but evidence of how these communities shape microbial co-occurrence and change phosphorus (P) correlated microbial interactions is still lacking. In this study, a random matrix theory-based network analysis of 16S rRNA genes was used to identify bacterial networks associated with the peanut rhizosphere and bulk in four long-term fertilization schemes. High-throughput sequencing data revealed that with the decrease of rhizosphere bacterial diversity, its positive covariation in the network increased, indicating stronger commensal and mutual assemblage with less functional redundancy in the rhizosphere. P availability is the major variable modulating the peanut rhizosphere microbial community in acidic soil. Two strongly positive P-correlated rhizosphere OTUs (closely related to Chitinophaga pinensis and Nitrospira moscoviensis), which have the potential to promote carbon-phosphorus and nitrogen-phosphorus synergistic conversion, respectively, were used to investigate the advantages of manure amendment in promoting rhizosphere P cycling in peanut planting agrosystems.

Nitrite-oxidizing bacteria (NOB) are vital players in the global nitrogen cycle that convert nitrite to nitrate during the second step of nitrification. Within this functional guild, members of the genus Nitrospira are most widespread, phylogenetically diverse, and physiologically versatile, and they drive nitrite oxidation in many natural and engineered ecosystems. Despite their ecological and biotechnological importance, our understanding of their energy metabolism is still limited. A major bottleneck for a detailed biochemical characterization of Nitrospira is biomass production, since they are slow-growing and fastidious microorganisms. In this study, we cultivated Nitrospira moscoviensis under nitrite-oxidizing conditions in a continuous stirred tank reactor (CSTR) system. This cultivation setup enabled accurate control of physicochemical parameters and avoided fluctuating levels of their energy substrate nitrite, thus ensuring constant growth conditions and furthermore allowing continuous biomass harvesting. Transcriptomic analyses under these conditions supported the predicted core metabolism of N. moscoviensis, including expression of all proteins required for carbon fixation via the reductive tricarboxylic acid cycle, assimilatory nitrite reduction, and the complete respiratory chain. Here, simultaneous expression of multiple copies of respiratory complexes I and III suggested functional differentiation. The transcriptome also indicated that the previously assumed membrane-bound nitrite oxidoreductase (NXR), the enzyme catalyzing nitrite oxidation, is formed by three soluble subunits. Overall, the transcriptomic data greatly refined our understanding of the metabolism of Nitrospira. Moreover, the application of a CSTR to cultivate Nitrospira is an important foundation for future proteomic and biochemical characterizations, which are crucial for a better understanding of these fascinating microorganisms.

The information about the impact of copper pipes on the growth of Legionella pneumophila in premise plumbing is controversial. For this reason, pipe segments of copper, stainless steel (SS), mild steel (MS), polyethylene, chlorinated polyvinylchloride (CPVC) and glass (controls) were exposed to intermittently flowing (20 min stagnation time) nonchlorinated tap water of 37 °C or 16 °C (ambient temperature) during six months to study the impact of metals on biofilm formation and growth of L. pneumophila. Biofilm concentrations (BfC, measured as ATP) on copper were 3 (at 37 °C) to 6 (at 16 °C) times higher than on SS. The maximum colony counts of L. pneumophila on the materials tested at 37 °C showed a quadratic relationship with the associated BfCs, with highest values on copper and MS. The average Cu concentration on the glass control of copper (glass-copper) was more than two log units lower than the Fe concentration on glass-MS, suggesting that copper released less corrosion by-products than MS. The release of corrosion by-products with attached biomass from MS most likely enhanced biofilm formation on glass-MS. Cloning and 16S RNA gene sequence analysis of the predominating biofilm bacteria revealed that an uncultured Xanthobacteraceae bacterium and Reyranella accounted for 75% of the bacterial community on copper at 37 °C. The nitrite-oxidizing Nitrospira moscoviensis, which can also utilize hydrogen (H2) and formate, accounted for >50% of the bacterial abundance in the biofilms on MS and glass-MS at 37 °C. The predominating presence of the strictly anaerobic non-fermentative Fe(III)-reducing Geobacter and the Fe(II)-oxidizing Gallionella on MS exposed to tap water of 16 °C indicated anoxic niches and the availability of H2, low molecular weight carboxylic acids (LMWCAs) and Fe(II) at the MS surface. LMWCAs likely also promoted bacterial growth on copper, but the release mechanisms from natural organic matter at the surface of corroding metals are unclear. The effects of water stagnation time and flow dynamics on biofilm formation on copper requires further investigation.

Rapid sand biofiltration (RSBF) is widely used for the removal of contaminants from drinking water treatment systems. Biofilm microbiomes in the biofilter media play essential roles in biotransformation of contaminants, but is not comprehensively understood. This study reports on Mn(II) oxidation and the core microbiomes in magnetite sand RSBF (MagS-RSBF) and manganese sand RSBF (MnS-RSBF). MnS-RSBF showed a relatively higher Mn(II) removal rate (40-91.2%) than MagS-RSBF during the start-up. MagS-RSBF and MnS-RSBF had similar Mn(II) removal rates (94.13% and 99.16%) over stable operation for 80 days. Mn(II) removal rates at different depths in the MnS-RSBF reactor significantly changed with operation time, and the filter in the upper layer of MnS-RSBF made the largest contribution to Mn(II) oxidation once operation had stabilized. PacBio single molecule sequencing of full-length 16S rRNA gene indicated that biofilter medium had a significant impact on the core microbiomes of the biofilms from the two biofilters. The magnetite sand biofilter facilitated the enrichment of Mn(II)-oxidizing biofilms. The dominant populations consisted of Pedomicrobium, Pseudomonas, and Hyphomicrobium in the RSBF, which have been affiliated with putative manganese-oxidizing bacteria (MnOB). The relative abundance of Pedomicrobium manganicum increased with operation time in both RSBF reactors. In addition, Nordella oligomobilis and Derxia gummosa were statistically correlated with Mn(II) oxidation. Species-species co-occurrence networks indicated that the microbiome of MnS-RSBF had more complex correlations than that of MagS-RSBF, implying that biofilter medium substantially shaped the microbial community in the RSBF. Hyphomicrobium and nitrite-oxidizing Nitrospira moscoviensis were positively correlated. The core microbiomes' composition of both RSBF reactors converged over operation time. A hybrid biofilter medium with magnetite sand and manganese sand may therefore be best in rapid sand filtration for Mn(II) oxidation.

Freshwater mussel assemblages of the Upper Mississippi River (UMR) sequester tons of ammonia- and urea-based biodeposits each day and aerate sediment through burrowing activities, thus creating a unique niche for nitrogen (N) cycling microorganisms. This study explored how mussels impact the abundance of N-cycling species with an emphasis on Candidatus Nitrospira inopinata, the first microorganism known to completely oxidize ammonia (comammox) to nitrate. This study used metagenomic shotgun sequencing of genomic DNA to compare nitrogen cycling species in sediment under a well-established mussel assemblage and in nearby sediment without mussels. Metagenomic reads were aligned to the prokaryotic RefSeq non-redundant protein database using BLASTx, taxonomic binning was performed using the weighted lowest common ancestor algorithm, and protein-coding genes were categorized by metabolic function using the SEED subsystem. Linear discriminant analysis (LDA) effect sizes were used to determine which metagenomes and metabolic features explained the most differences between the mussel habitat sediment and sediment without mussels. Of the N-cycling species deemed differentially abundant, Nitrospira moscoviensis and “Candidatus Nitrospira inopinata” were responsible for creating a distinctive N-cycling microbiome in the mussel habitat sediment. Further investigation revealed that comammox Nitrospira had a large metabolic potential to degrade mussel biodeposits, as evidenced the top ten percent of protein-coding genes including the cytochrome c-type biogenesis protein required for hydroxylamine oxidation, ammonia monooxygenase, and urea decomposition SEED subsystems. Genetic marker analysis of these two Nitrospira taxons suggested that N. moscoviensis was most impacted by diverse carbon metabolic processes while “Candidatus Nitrospira inopinata” was most distinguished by multidrug efflux proteins (AcrB), NiFe hydrogenase (HypF) used in hydrogen oxidation and sulfur reduction coupled reactions, and a heme chaperone (CcmE). Furthermore, our research suggests that comammox and NOB Nitrospira likely coexisted by utilizing mixotrophic metabolisms. For example, “Candidatus Nitrospira inopinata” had the largest potentials for ammonia oxidation, nitrite reduction with NirK, and hydrogen oxidation, while NOB Nitrospira had the greatest potential for nitrite oxidation, and nitrate reduction possibly coupled with formate oxidation. Overall, our results suggest that this mussel habitat sediment harbors a niche for NOB and comammox Nitrospira, and ultimately impacts N-cycling in backwaters of the UMR.

The genomes of many bacteria that participate in nitrogen cycling through the process of nitrification contain putative genes associated with acyl-homoserine lactone (AHL) quorum sensing (QS). AHL QS or bacterial cell-cell signaling is a method of bacterial communication and gene regulation and may be involved in nitrogen oxide fluxes or other important phenotypes in nitrifying bacteria. Here, we carried out a broad survey of AHL production in nitrifying bacteria in three steps. First, we analyzed the evolutionary history of AHL synthase and AHL receptor homologs in sequenced genomes and metagenomes of nitrifying bacteria to identify AHL synthase homologs in ammonia-oxidizing bacteria (AOB) of the genus Nitrosospira and nitrite-oxidizing bacteria (NOB) of the genera Nitrococcus , Nitrobacter , and Nitrospira . Next, we screened cultures of both AOB and NOB with uncharacterized AHL synthase genes and AHL synthase-negative nitrifiers by a bioassay. Our results suggest that an AHL synthase gene is required for, but does not guarantee, cell density-dependent AHL production under the conditions tested. Finally, we utilized mass spectrometry to identify the AHLs produced by the AOB Nitrosospira multiformis and Nitrosospira briensis and the NOB Nitrobacter vulgaris and Nitrospira moscoviensis as N -decanoyl- l -homoserine lactone (C 10 -HSL), N -3-hydroxy-tetradecanoyl- l -homoserine lactone (3-OH-C 14 -HSL), a monounsaturated AHL (C 10:1 -HSL), and N -octanoyl- l -homoserine lactone (C 8 -HSL), respectively. Our survey expands the list of AHL-producing nitrifiers to include a representative of Nitrospira lineage II and suggests that AHL production is widespread in nitrifying bacteria. IMPORTANCE Nitrification, the aerobic oxidation of ammonia to nitrate via nitrite by nitrifying microorganisms, plays an important role in environmental nitrogen cycling from agricultural fertilization to wastewater treatment. The genomes of many nitrifying bacteria contain genes associated with bacterial cell-cell signaling or quorum sensing (QS). QS is a method of bacterial communication and gene regulation that is well studied in bacterial pathogens, but less is known about QS in environmental systems. Our previous work suggested that QS might be involved in the regulation of nitrogen oxide gas production during nitrite metabolism. This study characterized putative QS signals produced by different genera and species of nitrifiers. Our work lays the foundation for future experiments investigating communication between nitrifying bacteria, the purpose of QS in these microorganisms, and the manipulation of QS during nitrification.

Microorganisms in groundwater play an important role in aquifer biogeochemical cycles and water quality. However, the mechanisms linking the functional diversity of microbial populations and the groundwater physico-chemistry are still not well understood due to the complexity of interactions between surface and subsurface. Within the framework of Hainich (north-western Thuringia, central Germany) Critical Zone Exploratory of the Collaborative Research Centre AquaDiva, we used the relative abundances of phospholipid-derived fatty acids (PLFAs) to link specific biochemical markers within the microbial communities to the spatio-temporal changes of the groundwater physico-chemistry. The functional diversities of the microbial communities were mainly correlated with groundwater chemistry, including dissolved O2, Fet and NH4+ concentrations. Abundances of PLFAs derived from eukaryotes and potential nitrite-oxidizing bacteria (11Me16:0 as biomarker for Nitrospira moscoviensis) were high at sites with elevated O2 concentration where groundwater recharge supplies bioavailable substrates. In anoxic groundwaters more rich in Fet, PLFAs abundant in sulfate-reducing bacteria (SRB), iron-reducing bacteria and fungi increased with Fet and HCO3− concentrations, suggesting the occurrence of active iron reduction and the possible role of fungi in meditating iron solubilization and transport in those aquifer domains. In more NH4+-rich anoxic groundwaters, anammox bacteria and SRB-derived PLFAs increased with NH4+ concentration, further evidencing the dependence of the anammox process on ammonium concentration and potential links between SRB and anammox bacteria. Additional support of the PLFA-based bacterial communities was found in DNA- and RNA-based Illumina MiSeq amplicon sequencing of bacterial 16S rRNA genes, which showed high predominance of nitrite-oxidizing bacteria Nitrospira, e.g. Nitrospira moscoviensis, in oxic aquifer zones and of anammox bacteria in more NH4+-rich anoxic groundwater. Higher relative abundances of sequence reads in the RNA-based datasets affiliated with iron-reducing bacteria in more Fet-rich groundwater supported the occurrence of active dissimilatory iron reduction. The functional diversity of the microbial communities in the biogeochemically distinct groundwater assemblages can be largely attributed to the redox conditions linked to changes in bioavailable substrates and input of substrates with the seepage. Our results demonstrate the power of complementary information derived from PLFA-based and sequencing-based approaches.

Annular reactors (ARs) were used to study biofilm community succession and provide ecological insight during nitrification arrestment through simultaneously increasing monochloramine (NH2Cl) and chlorine to nitrogen mass ratios, resulting in four operational periods (I-IV). Analysis of 16S rRNA-encoding gene sequence reads (454-pyrosequencing) examined viable and total biofilm communities and found total samples were representative of the underlying viable community. Bacterial community structure showed dynamic changes corresponding with AR operational parameters. Period I (complete nitrification and no NH2Cl residual) was dominated by Bradyrhizobium (total cumulative distribution: 38%), while environmental Legionella-like phylotypes peaked (19%) during Period II (complete nitrification and minimal NH2Cl residual). Nitrospira moscoviensis (nitrite-oxidizing bacteria) was detected in early periods (2%) but decreased to0.02% in later periods, corresponding to nitrite accumulation. Methylobacterium (19%) and members of Nitrosomonadaceae (42%) dominated Period III (complete ammonia and partial nitrite oxidation and low NH2Cl residual). An increase in Afipia (haloacetic acid-degrading bacteria) relative abundance (2% to 42%) occurred during Period IV (minimal nitrification and moderate to high NH2Cl residual). Microbial community and operational data provided no evidence of taxa-time relationship, but rapid community transitions indicated that the system had experienced ecological regime shifts to alternative stable states.

Despite its importance in the nitrogen cycle, little is known about nitrite oxidation at high temperatures. To bridge this gap, enrichment cultures were inoculated with sediment slurries from a variety of geothermal springs. While nitrite-oxidizing bacteria (NOB) were successfully enriched from seven hot springs located in US Great Basin, south-western China, and Armenia at ≤ 57.9 °C, all attempts to enrich NOB from10 hot springs at ≥ 61 °C failed. The stoichiometric conversion of nitrite to nitrate, chlorate sensitivity, and sensitivity to autoclaving all confirmed biological nitrite oxidation. Regardless of origin, all successful enrichments contained organisms with high 16S rRNA gene sequence identity (≥ 97%) with Nitrospira calida. In addition, Armenian enrichments also contained close relatives of Nitrospira moscoviensis. Physiological properties of all enrichments were similar, with a temperature optimum of 45-50 °C, yielding nitrite oxidation rates of 7.53 ± 1.20 to 23.0 ± 2.73 fmoles cell(-1) h(-1), and an upper temperature limit between 60 and 65 °C. The highest rates of NOB activity occurred with initial NO2 - concentrations of 0.5-0.75 mM; however, lower initial nitrite concentrations resulted in shorter lag times. The results presented here suggest a possible upper temperature limit of 60-65 °C for Nitrospira and demonstrate the wide geographic range of Nitrospira species in geothermal environments.

Nitrite oxidation is an important step in nitrogen removal in biological wastewater treatment plants. Nitrite-oxidizing bacteria (NOB) involved in nitrite oxidation in wastewater treatment plants are known to belong to the genus Nitrobacter or the genus Nitrospira. Previously, the physiological and biochemical properties of NOB were mainly investigated using Nitrobacter strains because they were easily cultured in the laboratory. However, cultivation-independent molecular methods (5, 10, 11, 27) and immunological techniques (3) indicated that members of the genus Nitrospira and not Nitrobacter species were dominant NOB in aquaria and full-scale wastewater treatment plants. The comparative analysis of 16S rRNA gene sequences revealed that the genus Nitrospira was classified into four different phylogenetic lineages, and sublineages I and II play a key role as nitrite oxidizers in wastewater treatment plants (6). Because Nitrospira-like bacteria in nitrifying activated sludge or biofilms are fastidious and slow-growing organisms, their physiological properties have been investigated by molecular-based approaches (6, 18, 21, 28). However, high enrichment or pure culture is required to determine detailed biochemical properties and genomic information. Recently, “Candidatus Nitrospira defluvii” belonging to sublineage I was enriched from activated sludge, and its physiological properties were characterized (23). Moreover, the complete genome of “Ca. Nitrospira defluvii” allowed insight into the key metabolic pathways of Nitrospira and its evolution (14). Meanwhile, no Nitrospira within sublineage II has been isolated from wastewater treatment plants so far. The major members of the genus Nitrospira sublineage II in activated sludge are known to be phylogenetically distant from Nitrospira moscoviensis, which was isolated from an urban heating system in Moscow and classified as sublineage II (7). Here, we isolated pure cultures belonging to sublineage II from activated sludge. Previously, we enriched sublineage II Nitrospira from activated sludge using a continuous feeding bioreactor (9), but could not obtain pure cultures partly because growing Nitrospira cells tended to form micro-colonies. In this study, we developed a new isolation method, which enabled selective separation of Nitrospira micro-colonies from the enrichment culture via a cell-sorting system. As a result, we achieved pure culture of sublineage II Nitrospira from activated sludge.

Total microbial community structure, and particularly nitrifying communities inhabiting five different small drinking water networks characterized with different water physical and chemical parameters was investigated, using cultivation-based methods and sequence aided Terminal Restriction Fragment Length Polymorphism (T-RFLP) analysis. Ammonium ion, originated from well water, was only partially oxidized via nitrite to nitrate in the drinking water distribution systems. Nitrification occurred at low ammonium ion concentration (27–46 μM), relatively high pH (7.6–8.2) and over a wide range of dissolved oxygen concentrations (0.4–9.0 mg L −1 ). The nitrifying communities of the distribution systems were characterized by variable most probable numbers (2 × 10 2 –7.1 × 10 4 MPN L −1 ) and probably originated from the non-treated well water. The sequence aided T-RFLP method revealed that ammonia-oxidizing microorganisms and nitrite-oxidizing Bacteria ( Nitrosomonas oligotropha , Nitrosopumilus maritimus, and Nitrospira moscoviensis, ‘ Candidatus Nitrospira defluvii’) were present in different ratios in the total microbial communities of the distinct parts of the water network systems. The nitrate generated by nitrification was partly utilized by nitrate-reducing (and denitrifying) Bacteria, present in low MPN and characterized by sequence aided T-RFLP as Comamonas sp. and Pseudomonas spp. Different environmental factors, like pH, chemical oxygen demand, calculated total inorganic nitrogen content (moreover nitrite and nitrate concentration), temperature had important effect on the total bacterial and archaeal community distribution.

This study analysed the nitrifier community in the biofilter of a zero discharge, recirculating aquaculture system (RAS) for the production of marine shrimp in a low density (low ammonium production) system. The ammonia-oxidizing populations were examined by targeting 16S rRNA and amoA genes of ammonia-oxidizing bacteria (AOB) and archaea (AOA). The nitrite-oxidizing bacteria (NOB) were investigated by targeting the 16S rRNA gene. Archaeal amoA genes were more abundant in all compartments of the RAS than bacterial amoA genes. Analysis of bacterial and archaeal amoA gene sequences revealed that most ammonia oxidizers were related to Nitrosomonas marina and Nitrosopumilus maritimus . The NOB detected were related to Nitrospira marina and Nitrospira moscoviensis, and Nitrospira marina -type NOB were more abundant than N. moscoviensis -type NOB. Water quality and biofilm attachment media played a role in the competitiveness of AOA over AOB and Nitrospira marina- over N. moscoviensis- type NOB.

The effects of growth type, including attached growth, suspended growth, and combined growth, on the characteristics of communities of ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) were studied in three lab-scale Anaerobic/Anoxicm-Oxicn (AmOn) systems. These systems amplified activated sludge, biofilms, and a mixture of activated sludge and biofilm (AS-BF). Identical inocula were adopted to analyze the selective effects of mixed growth patterns on nitrifying bacteria. Fluctuations in the concentration of nitrifying bacteria over the 120 days of system operation were analyzed, as was the composition of nitrifying bacterial community in the stabilized stage. Analysis was conducted using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) and real-time PCR. According to the DGGE patterns, the primary AOB lineages were Nitrosomonas europaea (six sequences), Nitrosomonas oligotropha (two sequences), and Nitrosospira (one sequence). The primary subclass of NOB community was Nitrospira, in which all identified sequences belonged to Nitrospira moscoviensis (14 sequences). Nitrobacter consisted of two lineages, namely Nitrobacter vulgaris (three sequences) and Nitrobacter alkalicus (two sequences). Under identical operating conditions, the composition of nitrifying bacterial communities in the AS-BF system demonstrated significant differences from those in the activated sludge system and those in the biofilm system. Major varieties included several new, dominant bacterial sequences in the AS-BF system, such as N. europaea and Nitrosospira and a higher concentration of AOB relative to the activated sludge system. However, no similar differences were discovered for the concentration of the NOB population. A kinetic study of nitrification demonstrated a higher maximum specific growth rate of mixed sludge and a lower half-saturation constant of mixed biofilm, indicating that the AS-BF system maintained relatively good nitrifying ability.

Oxidizing hydrogen in place of nitrite Microorganisms are important drivers of Earth's nitrogen cycle. Many of the organisms responsible for mediating the reactions of one phase of nitrogen to another are thought to be ecologic specialists. Using a combination of genomic and experimental analyses, Koch et al. show that Nitrospira moscoviensis , a member of a widely distributed genus of nitrite-oxidizing bacteria, can oxidize hydrogen instead of nitrite to support growth when oxygen is present. Not only does this ecologic flexibility suggest a broader distribution of these organisms in natural settings, but they may be important in engineered environments as well. Science , this issue p. 1052

Nitrospira are barely studied and mostly uncultured nitrite-oxidizing bacteria, which are, according to molecular data, among the most diverse and widespread nitrifiers in natural ecosystems and biological wastewater treatment. Here, environmental genomics was used to reconstruct the complete genome of “ Candidatus Nitrospira defluvii” from an activated sludge enrichment culture. On the basis of this first-deciphered Nitrospira genome and of experimental data, we show that Ca . N. defluvii differs dramatically from other known nitrite oxidizers in the key enzyme nitrite oxidoreductase (NXR), in the composition of the respiratory chain, and in the pathway used for autotrophic carbon fixation, suggesting multiple independent evolution of chemolithoautotrophic nitrite oxidation. Adaptations of Ca . N. defluvii to substrate-limited conditions include an unusual periplasmic NXR, which is constitutively expressed, and pathways for the transport, oxidation, and assimilation of simple organic compounds that allow a mixotrophic lifestyle. The reverse tricarboxylic acid cycle as the pathway for CO 2 fixation and the lack of most classical defense mechanisms against oxidative stress suggest that Nitrospira evolved from microaerophilic or even anaerobic ancestors. Unexpectedly, comparative genomic analyses indicate functionally significant lateral gene-transfer events between the genus Nitrospira and anaerobic ammonium-oxidizing planctomycetes, which share highly similar forms of NXR and other proteins reflecting that two key processes of the nitrogen cycle are evolutionarily connected.

Ciliated protists are important predators of bacteria in many aquatic habitats, including sediments. Since, many biochemical transformations within the nitrogen cycle are performed by bacteria, ciliates could have an indirect impact on the nitrogen cycle through selective grazing on nitrogen-transforming bacteria. As a case study, we examined ciliate grazing on nitrifying bacteria of the genera Nitrosomonas and Nitrospira. All experiments were designed as in vitro-experiments with cultures of different bacteria and ciliate species. The nitrifying bacteria used in our experiments were Nitrosomonas europaea [Winogradsky 1892] and Nitrospira moscoviensis [Ehrich 2001]. The ciliates comprised of four species that are known as efficient bacterivores and common members of the protist community in aquatic systems: Paramecium aurelia [Muller 1773], Euplotes octocarinatus [Carter 1972], Tetrahymena pyriformis [Ehrenberg 1830] and Cyclidium glaucoma [Muller 1786]. Our experimental approach, using a combination of DAPI and FISH staining, was successful in allowing the observation of ingestion of specific bacteria and their detection within ciliate food vacuoles. However, the ciliates in this study showed no significant selective grazing. No food preferences for a any bacterial taxon or any size class or morphotype were detected. Correlation with time between ciliate abundance and bacterial abundance or biovolume, using log transformed growth rates of ciliates and bacteria, showed no significant results. On the bacterial side, neither an active defence mechanism of the nitrifying bacteria against ciliate grazing, such as changes in morphology, nor competition for resources were observed. These results suggest that in our in vitro-experiments grazing by ciliates has no influence on abundance and growth of nitrifying bacteria and nitrification.

Batch tests were carried out with Nitrospira in mixed and pure cultures using concentrations of free ammonia widely believed to be inhibitory to nitrite-oxidizing organisms. The mixed culture batch tests were conducted with mixed liquor from a bench scale completely stirred tank reactor (CSTR) treating a synthetic wastewater having a low C:N ratio. Nitrospira were confirmed as the dominant nitrite oxidizers via RNA slot blotting. Nitrospira moscoviensis were used for the pure culture trials. The results from this study suggest that free ammonia (NH3-N) concentrations of up to 10 mg/L were not inhibitory to Nitrospira either in situ or in pure culture. Key words: fatty acid methyl esters, free ammonia inhibition, molecular methods, nitrification, nitrite, Nitrobacter, Nitrospira, nitrous acid, nitrous oxide, RNA slot blotting, wastewater treatment.

In a model drinking water distribution system characterized by a low assimilable organic carbon content (Proteobacteria and Bacteriodetes , whereas independently of cultivation, bacteria from 12 phyla were detected in this system. These included Acidobacteria , Nitrospirae , Planctomycetes , and Verrucomicrobia , some of which have never been identified in drinking water previously. A cluster analysis of the population profiles from the individual samples divided biofilms and bulk water samples into separate clusters ( P = 0.027). Bacteria associated with Nitrospira moscoviensis were found in all samples and encompassed 39% of the sequenced clones in the bulk water and 25% of the biofilm community. The close association with Nitrospira suggested that a large part of the population had an autotrophic metabolism using nitrite as an electron donor. To test this hypothesis, nitrite was added to biofilm and bulk water samples, and the utilization was monitored during 15 days. A first-order decrease in nitrite concentration was observed for all samples with a rate corresponding to 0.5 × 10 5 to 2 × 10 5 nitrifying cells/ml in the bulk water and 3 × 10 5 cells/cm 2 on the pipe surface. The finding of an abundant nitrite-oxidizing microbial population suggests that nitrite is an important substrate in this system, potentially as a result of the low assimilable organic carbon concentration. This finding implies that microbial communities in water distribution systems may control against elevated nitrite concentrations but also contain large indigenous populations that are capable of assisting the depletion of disinfection agents like chloramines.

The microbial communities associated with filter materials in closed recirculated systems of carp and goldfish were examined by the clone library method of 16S rRNA gene. The bacterial cells were efficiently recovered by the combination of six washing treatment and swabbing. The bacterial density on pebbles of well-conditioned recirculating water systems was 1.1×107 cells g−1 in the carp-rearing tank and 1.9×108 cells g−1 in the goldfish-rearing aquarium. The 91 cloned rDNA sequences were occupied by members within Alphaproteobacteria (52 clones belonging to 19 taxa), Betaproteobacteria (14 clones belonging to 8 taxa), Nitrospira (8 clones belonging to 1 taxon), Actinobacteria (6 clones belonging to 4 taxa), Bacilli (3 clones belonging to 3 taxa), Gammaproteobacteria (3 clones belonging to 3 taxa), Planctomycetacia (3 clones belonging to 3 taxa) and Sphingobacteria (2 clones belonging to 1 taxon). However, only three taxa, which are closely related to Hyphomicrobium denitrificans, Rhodovulum euryhalinum and Nitrospira moscoviensis, were commonly detected in both carp- and goldfish-rearing systems. These results reveal that there are great differences in both bacterial density and composition between the two different systems, and that the clone library constructed from carp-rearing tank is characterized by as many as 29 taxa of bacteria, and that those from goldfish-rearing aquarium is characterized by Hyphomicrobium facilis with occurrence of 42%. These results mean that at least 36% of total clones could utilize the inorganic nitrogen, NH4+, NO2− or NO3−, suggesting that they concern the dynamics of nitrogen in fish-rearing facilities equipped with the recirculating system.

The nature of unusual aquatic microbial formations in flooded passages of cave systems in the Nullarbor region of Australia was investigated using electron microscopy and DNA analysis. The caves are located in a semiarid region but intersect the watertable at depths of approximately 100 m below the surface. Throughout submerged portions of the caves divers have noted the presence of unusual microbial formations. These 'microbial mantles' comprise sheets or tongues of mucoid material in which small crystals are embedded. Examination of the biomass revealed it to be primarily composed of densely packed, unbranched filaments, together with spherical-, rod- and spiral-shaped cells, and microcrystals of calcite in a mucoid matrix. Molecular phylogenetic analysis of the community structure revealed approximately 12% of clones showed high similarity to autotrophic nitrite-oxidizing bacteria (Nitrospira moscoviensis). The remainder of the clones exhibited a high proportion of phylogenetically novel sequence types. Chemical analysis of water samples revealed high levels of sulphate and nitrate together with significant nitrite. The community structure, the presence of nitrite in the water, and the apparent absence of aquatic macrofauna, suggest these microbial structures may represent biochemically novel, chemoautotrophic communities dependent on nitrite oxidation.

Summary The fatty acid profiles of all described species of the nitrite-oxidizing genera Nitrobacter , Nitrococcus , Nitrospina and Nitrospira were analyzed. The four genera had distinct profiles, which can be used for the differentiation and allocation of new isolates to these genera. The genus Nitrobacter is characterized by vaccenic acid as the main compound with up to 92% of the fatty acids and the absence of hydroxy fatty acids. The genus Nitrococcus showed cis -9-hexadecenoic acid, hexadecanoic acid and vaccenic acid as main parts. Small amounts of 3-hydroxy-dodecanoic acid were detected. The genus Nitrospina possessed tetradecanoic acid and cis -9-hexadecenoic acid as main compounds, also 3-hydroxy-hexadecanoic acid was detected for this genus. The genus Nitrospira showed a pattern with more variations among the two described species. These organisms are characterized by the cis -7 and cis -11-isomers of hexadecenoic acid. For Nitrospira moscoviensis a specific new fatty acid was found, which represented the major constituent in the fatty acid profiles of autotrophically grown cultures. It was identified as 11-methyl-hexadecanoic acid. Since this compound is not known for other bacterial taxa, it represents a potential lipid marker for the detection of Nitrospira moscoviensis relatives in enrichment cultures and environmental samples. A cluster analysis of the fatty acid profiles is in accordance with 16S rRNA sequence-based phylogeny of the nitrite-oxidizing bacteria.

A competitive immunosensor using a monoclonal antibody has been developed for the enumeration of Nitrobacter in activated sludge and other environmental samples. Its cross-reactivity was tested against a number of bacterial strains and isolates. All strains of the nitrite-oxidising genera Nitrobacter and Nitrococcus reacted strongly with the monoclonal antibody. The nitrite-oxidising Nitrospira moscoviensis, as well as the ammonia oxidising bacteria and the heterotrophic bacteria tested, did not show any affinity towards the antibody in the immunosensor. The numbers of Nitrobacter were analysed in sludge samples from several wastewater treatment plants in Sweden. Detectable amounts were found in all samples. This study shows the adequacy of using this immunosensor for the enumeration of Nitrobacter in natural environments.

The change of activity and abundance of Nitrosospira and Nitrospira spp. along a bulk water gradient in a nitrifying fluidized bed reactor was analyzed by a combination of microsensor measurements and fluorescence in situ hybridization. Nitrifying bacteria were immobilized in bacterial aggregates that remained in fixed positions within the reactor column due to the flow regimen. Nitrification occurred in a narrow zone of 100 to 150 μm on the surface of these aggregates, the same layer that contained an extremely dense community of nitrifying bacteria. The central part of the aggregates was inactive, and significantly fewer nitrifiers were found there. Under conditions prevailing in the reactor, i.e., when ammonium was limiting, ammonium was completely oxidized to nitrate within the active layer of the aggregates, the rates decreasing with increasing reactor height. To analyze the nitrification potential, profiles were also recorded in aggregates subjected to a short-term incubation under elevated substrate concentrations. This led to a shift in activity from ammonium to nitrite oxidation along the reactor and correlated well with the distribution of the nitrifying population. Along the whole reactor, the numbers of ammonia-oxidizing bacteria decreased, while the numbers of nitrite-oxidizing bacteria increased. Finally, volumetric reaction rates were calculated from microprofiles and related to cell numbers of nitrifying bacteria in the active shell. Therefore, it was possible for the first time to estimate the cell-specific activity of Nitrosospira spp. and hitherto-uncultured Nitrospira -like bacteria in situ.

A sequencing batch reactor (SBR) was operated to selectively grow a nitrite oxidising microbial community and was called the nitrite oxidising SBR (NOSBR). The nitrite oxidising characteristics of the reactor biomass were studied as well as the microbial composition. Molecular biological methods of clone libraries were used to evaluate the microorganisms in both the seed sludge and in the NOSBR sludge. We have found that the nitrite oxidation in the NOSBR was due the presence of bacteria from the Nitrospira phylum and not because of the presence of Nitrobacter which were in very low numbers in the NOSBR and not detected in the seed sludge. We hypothesize that the unknown nitrite oxidising bacteria in wastewater treatment plants are a range of species related to Nitrospira moscoviensis. A suite of primers were developed from the clone sequence data and used in a diagnostic polymerase chain reaction to prove the presence of these novel nitrite oxidisers in a range of full scale and laboratory scale activated sludge plants.

The ammonia-oxidizing and nitrite-oxidizing bacterial populations occurring in the nitrifying activated sludge of an industrial wastewater treatment plant receiving sewage with high ammonia concentrations were studied by use of a polyphasic approach. In situ hybridization with a set of hierarchical 16S rRNA-targeted probes for ammonia-oxidizing bacteria revealed the dominance of Nitrosococcus mobilis -like bacteria. The phylogenetic affiliation suggested by fluorescent in situ hybridization (FISH) was confirmed by isolation of N. mobilis as the numerically dominant ammonia oxidizer and subsequent comparative 16S rRNA gene (rDNA) sequence and DNA-DNA hybridization analyses. For molecular fine-scale analysis of the ammonia-oxidizing population, a partial stretch of the gene encoding the active-site polypeptide of ammonia monooxygenase ( amoA ) was amplified from total DNA extracted from ammonia oxidizer isolates and from activated sludge. However, comparative sequence analysis of 13 amoA clone sequences from activated sludge demonstrated that these sequences were highly similar to each other and to the corresponding amoA gene fragments of Nitrosomonas europaea Nm50 and the N. mobilis isolate. The unexpected high sequence similarity between the amoA gene fragments of the N. mobilis isolate and N. europaea indicates a possible lateral gene transfer event. Although a Nitrobacter strain was isolated, members of the nitrite-oxidizing genus Nitrobacter were not detectable in the activated sludge by in situ hybridization. Therefore, we used the rRNA approach to investigate the abundance of other well-known nitrite-oxidizing bacterial genera. Three different methods were used for DNA extraction from the activated sludge. For each DNA preparation, almost full-length genes encoding small-subunit rRNA were separately amplified and used to generate three 16S rDNA libraries. By comparative sequence analysis, 2 of 60 randomly selected clones could be assigned to the nitrite-oxidizing bacteria of the genus Nitrospira . Based on these clone sequences, a specific 16S rRNA-targeted probe was developed. FISH of the activated sludge with this probe demonstrated that Nitrospira -like bacteria were present in significant numbers (9% of the total bacterial counts) and frequently occurred in coaggregated microcolonies with N. mobilis .

A hierarchical set of five 16S rRNA-targeted oligonucleotide DNA probes for phylogenetically defined groups of autotrophic ammonia- and nitrite-oxidizing bacteria was developed for environmental and determinative studies. Hybridization conditions were established for each probe by using temperature dissociation profiles of target and closely related nontarget organisms to document specificity. Environmental application was demonstrated by quantitative slot blot hybridization and whole-cell hybridization of nitrifying activated sludge and biofilm samples. Results obtained with both techniques suggested the occurrence of novel populations of ammonia oxidizers. In situ hybridization experiments revealed that Nitrobacter and Nitrosomonas species occurred in clusters and frequently were in contact with each other within sludge flocs.

Oxidation of nitrite to nitrate in aquaria is typically attributed to bacteria belonging to the genus Nitrobacter which are members of the α subdivision of the class Proteobacteria . In order to identify bacteria responsible for nitrite oxidation in aquaria, clone libraries of rRNA genes were developed from biofilms of several freshwater aquaria. Analysis of the rDNA libraries, along with results from denaturing gradient gel electrophoresis (DGGE) on frequently sampled biofilms, indicated the presence of putative nitrite-oxidizing bacteria closely related to other members of the genus Nitrospira . Nucleic acid hybridization experiments with rRNA from biofilms of freshwater aquaria demonstrated that Nitrospira -like rRNA comprised nearly 5% of the rRNA extracted from the biofilms during the establishment of nitrification. Nitrite-oxidizing bacteria belonging to the α subdivision of the class Proteobacteria (e.g., Nitrobacter spp.) were not detected in these samples. Aquaria which received a commercial preparation containing Nitrobacter species did not show evidence of Nitrobacter growth and development but did develop substantial populations of Nitrospira -like species. Time series analysis of rDNA phylotypes on aquaria biofilms by DGGE, combined with nitrite and nitrate analysis, showed a correspondence between the appearance of Nitrospira -like bacterial ribosomal DNA and the initiation of nitrite oxidation. In total, the data suggest that Nitrobacter winogradskyi and close relatives were not the dominant nitrite-oxidizing bacteria in freshwater aquaria. Instead, nitrite oxidation in freshwater aquaria appeared to be mediated by bacteria closely related to Nitrospira moscoviensis and Nitrospira marina.

Uncultivated Nitrospira -like bacteria in different biofilm and activated-sludge samples were investigated by cultivation-independent molecular approaches. Initially, the phylogenetic affiliation of Nitrospira -like bacteria in a nitrifying biofilm was determined by 16S rRNA gene sequence analysis. Subsequently, a phylogenetic consensus tree of the Nitrospira phylum including all publicly available sequences was constructed. This analysis revealed that the genus Nitrospira consists of at least four distinct sublineages. Based on these data, two 16S rRNA-directed oligonucleotide probes specific for the phylum and genus Nitrospira , respectively, were developed and evaluated for suitability for fluorescence in situ hybridization (FISH). The probes were used to investigate the in situ architecture of cell aggregates of Nitrospira -like nitrite oxidizers in wastewater treatment plants by FISH, confocal laser scanning microscopy, and computer-aided three-dimensional visualization. Cavities and a network of cell-free channels inside the Nitrospira microcolonies were detected that were water permeable, as demonstrated by fluorescein staining. The uptake of different carbon sources by Nitrospira -like bacteria within their natural habitat under different incubation conditions was studied by combined FISH and microautoradiography. Under aerobic conditions, the Nitrospira -like bacteria in bioreactor samples took up inorganic carbon (as HCO 3 − or as CO 2 ) and pyruvate but not acetate, butyrate, and propionate, suggesting that these bacteria can grow mixotrophically in the presence of pyruvate. In contrast, no uptake by the Nitrospira -like bacteria of any of the carbon sources tested was observed under anoxic or anaerobic conditions.

Bacterial aggregates from a chemolithoautotrophic, nitrifying fluidized bed reactor were investigated with microsensors and rRNA-based molecular techniques. The microprofiles of O 2 , NH 4 + , NO 2 − , and NO 3 − demonstrated the occurrence of complete nitrification in the outer 125 μm of the aggregates. The ammonia oxidizers were identified as members of the Nitrosospira group by fluorescence in situ hybridization (FISH). No ammonia- or nitrite-oxidizing bacteria of the genus Nitrosomonas or Nitrobacter , respectively, could be detected by FISH. To identify the nitrite oxidizers, a 16S ribosomal DNA clone library was constructed and screened by denaturing gradient gel electrophoresis and selected clones were sequenced. The organisms represented by these sequences formed two phylogenetically distinct clusters affiliated with the nitrite oxidizer Nitrospira moscoviensis . 16S rRNA-targeted oligonucleotide probes were designed for in situ detection of these organisms. FISH analysis showed that the dominant populations of Nitrospira spp. and Nitrosospira spp. formed separate, dense clusters which were in contact with each other and occurred throughout the aggregate. A second, smaller, morphologically and genetically different population of Nitrospira spp. was restricted to the outer nitrifying zones.

A gram-negative, non-motile, non-marine, nitrite-oxidizing bacterium was isolated from an enrichment culture initiated with a sample from a partially corroded area of an iron pipe of a heating system in Moscow, Russia. The cells were 0.9–2.2 μm×0.2–0.4 μm in size. They were helical- to vibroid-shaped and often formed spirals with up to three turns 0.8–1.0 μm in width. The organism possessed an enlarged periplasmic space and lacked intracytoplasmic membranes and carboxysomes. The cells tended to excrete extracellular polymers, forming aggregates. The bacterium grew optimally at 39°C and pH 7.6–8.0 in a mineral medium with nitrite as sole energy source and carbon dioxide as sole carbon source. The optimal nitrite concentration was 0.35 mM. Nitrite was oxidized to nitrate stoichiometrically. The doubling time was 12 h in a mineral medium with 7.5 mM nitrite. The cell yield was low; only 0.9 mg protein/l was formed during oxidation of 7.5 mM nitrite. Under anoxic conditions, hydrogen was used as electron donor with nitrate as electron acceptor. Organic matter (yeast extract, meat extract, peptone) supported neither mixotrophic nor heterotrophic growth. At concentrations as low as 0.75 g organic matter/l or higher, growth of nitrite-oxidizing cells was inhibited. The cells contained cytochromes of theb- andc-type. The G+C content of DNA was 56.9±0.4 mol%. The chemolithoautotrophic nitrite-oxidizer differed from the terrestrial members of the genusNitrobacter with regard to morphology and substrate range and equaledNitrospira marina in both characteristics. The isolated bacterium is designated as a new species of the genusNitrospira. Comparative analysis of 16S rRNA gene sequences revealed a moderate phylogenetic relationship toNitrospira marina, leptospirilla,Thermodesulfovibrio yellowstonii, “Magnetobacterium bavaricum”, and the isolate OPI-2. Initial evidence is given that these organisms represent a new phylum of the domain bacteria.

A new chemolithotrophic nitrite-oxidizing bacterium, for which the name Nitrospira marina is proposed, was isolated from the Gulf of Maine. N. marina is a Gramnegative curved rod which may form spirals with 1 to 12 turns. Cells have a unique periplasmic space and lack intracytoplasmic membranes and carboxysomes. N. marina is an obligate chemolithotroph, but best growth is obtained in a mixotrophic medium. N. marina may be one of the most prevalent nitrite-oxidizing bacteria in some oceanic environments. Type strain is field with American Type Culture Collection (ATCC 43039).

The microbiology of the biomass from a nitrite-oxidizing sequencing batch reactor (NOSBR) fed with an inorganic salts solution and nitrite as the sole energy source that had been operating for 6 months was investigated by microscopy, by culture-dependent methods, and by molecular biological methods, and the seed sludge that was used to inoculate the NOSBR was investigated by molecular biological methods. The NOSBR sludge comprised a complex and diverse microbial community containing gram-negative and gram-positive rods, cocci, and filaments. By culture-dependent methods (i.e., micromanipulation and sample dilution and spread plate inoculation), 16 heterotrophs (6 gram positive and 10 gram negative) were identified in the NOSBR sludge (RC), but no autotrophs were isolated. 16S ribosomal DNA clone libraries of the two microbial communities revealed that the seed sludge (GC) comprised a complex microbial community dominated by Proteobacteria (29% beta subclass; 18% gamma subclass) and high G+C gram-positive bacteria (10%). Three clones (4%) were closely related to the autotrophic nitrite-oxidizer Nitrospira moscoviensis . The NOSBR sludge was overwhelmingly dominated by bacteria closely related to N. moscoviensis (89%). Two clone sequences were similar to those of the genus Nitrobacter . Near-complete insert sequences of eight RC and one GC N. moscoviensis clone were determined and phylogenetically analyzed. This is the first report of the presence of bacteria from the Nitrospira phylum in wastewater treatment systems, and it is hypothesized that these bacteria are the unknown nitrite oxidizers in these processes.

Nitrospira are a diverse group of nitrite-oxidizing bacteria and among the environmentally most widespread nitrifiers. However, they remain scarcely studied and mostly uncultured. Based on genomic and experimental data from Nitrospira moscoviensis representing the ubiquitous Nitrospira lineage II, we identified ecophysiological traits that contribute to the ecological success of Nitrospira. Unexpectedly, N. moscoviensis possesses genes coding for a urease and cleaves urea to ammonia and CO2. Ureolysis was not observed yet in nitrite oxidizers and enables N. moscoviensis to supply ammonia oxidizers lacking urease with ammonia from urea, which is fully nitrified by this consortium through reciprocal feeding. The presence of highly similar urease genes in Nitrospira lenta from activated sludge, in metagenomes from soils and freshwater habitats, and of other ureases in marine nitrite oxidizers, suggests a wide distribution of this extended interaction between ammonia and nitrite oxidizers, which enables nitrite-oxidizing bacteria to indirectly use urea as a source of energy. A soluble formate dehydrogenase lends additional ecophysiological flexibility and allows N. moscoviensis to use formate, with or without concomitant nitrite oxidation, using oxygen, nitrate, or both compounds as terminal electron acceptors. Compared with Nitrospira defluvii from lineage I, N. moscoviensis shares the Nitrospira core metabolism but shows substantial genomic dissimilarity including genes for adaptations to elevated oxygen concentrations. Reciprocal feeding and metabolic versatility, including the participation in different nitrogen cycling processes, likely are key factors for the niche partitioning, the ubiquity, and the high diversity of Nitrospira in natural and engineered ecosystems.