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PubMed1995

Amplification of 16S ribosomal RNA genes of autotrophic ammonia-oxidizing bacteria demonstrates the ubiquity of nitrosospiras in the environment.

Oligonucleotide sequences selected from the 16S rRNA genes of various species of ammonia-oxidizing bacteria were evaluated as specific PCR amplification primers and probes. The specificities of primer pairs for eubacterial, Nitrosospira and Nitrosomonas rRNA genes were established with sequence databases, and the primer pairs were used to amplify DNA from laboratory cultures and environmental samples. Eubacterial rRNA genes amplified from samples of soil and activated sludge hybridized with an oligonucleotide probe specific for Nitrosospira spp., but not with a Nitrosomonas-specific probe. Lakewater and sediment samples were analysed using a nested PCR technique in which eubacterial rRNA genes were subjected to a secondary amplification with Nitrosomonas or Nitrosospira specific primers. Again, the presence of Nitrosospira DNA, but not Nitrosomonas DNA, was detected and this was confirmed by hybridization of the amplified DNA with an internal oligonucleotide probe. Enrichments of lakewater and sediment samples, incubated for two weeks in the presence of ammonium, produced nitrite and were found to contain DNA from both Nitrosospira and Nitrosomonas as determined by nested PCR amplification and probing of 16S rRNA genes. This demonstrates that Nitrosospira spp. are widespread in the environment. The implications of the detection of Nitrosomonas DNA only after enrichment culture are discussed.

Ammonia↗
PubMed2001

Identification of bacteria responsible for ammonia oxidation in freshwater aquaria.

Culture enrichments and culture-independent molecular methods were employed to identify and confirm the presence of novel ammonia-oxidizing bacteria (AOB) in nitrifying freshwater aquaria. Reactors were seeded with biomass from freshwater nitrifying systems and enriched for AOB under various conditions of ammonia concentration. Surveys of cloned rRNA genes from the enrichments revealed four major strains of AOB which were phylogenetically related to the Nitrosomonas marina cluster, the Nitrosospira cluster, or the Nitrosomonas europaea-Nitrosococcus mobilis cluster of the beta subdivision of the class Proteobacteria. Ammonia concentration in the reactors determined which AOB strain dominated in an enrichment. Oligonucleotide probes and PCR primer sets specific for the four AOB strains were developed and used to confirm the presence of the AOB strains in the enrichments. Enrichments of the AOB strains were added to newly established aquaria to determine their ability to accelerate the establishment of ammonia oxidation. Enrichments containing the Nitrosomonas marina-like AOB strain were most efficient at accelerating ammonia oxidation in newly established aquaria. Furthermore, if the Nitrosomonas marina-like AOB strain was present in the original enrichment, even one with other AOB, only the Nitrosomonas marina-like AOB strain was present in aquaria after nitrification was established. Nitrosomonas marina-like AOB were 2% or less of the cells detected by fluorescence in situ hybridization analysis in aquaria in which nitrification was well established.

Ammonia↗
PubMed2006

Changes in the community structure and activity of betaproteobacterial ammonia-oxidizing sediment bacteria along a freshwater-marine gradient.

To determine whether the distribution of estuarine ammonia-oxidizing bacteria (AOB) was influenced by salinity, the community structure of betaproteobacterial ammonia oxidizers (AOB) was characterized along a salinity gradient in sediments of the Ythan estuary, on the east coast of Scotland, UK, by denaturant gradient gel electrophoresis (DGGE), cloning and sequencing of 16S rRNA gene fragments. Ammonia-oxidizing bacteria communities at sampling sites with strongest marine influence were dominated by Nitrosospira cluster 1-like sequences and those with strongest freshwater influence were dominated by Nitrosomonas oligotropha-like sequences. Nitrosomonas sp. Nm143 was the prevailing sequence type in communities at intermediate brackish sites. Diversity indices of AOB communities were similar at marine- and freshwater-influenced sites and did not indicate lower species diversity at intermediate brackish sites. The presence of sequences highly similar to the halophilic Nitrosomonas marina and the freshwater strain Nitrosomonas oligotropha at identical sampling sites indicates that AOB communities in the estuary are adapted to a range of salinities, while individual strains may be active at different salinities. Ammonia-oxidizing bacteria communities that were dominated by Nitrosospira cluster 1 sequence types, for which no cultured representative exists, were subjected to stable isotope probing (SIP) with 13C-HCO3-, to label the nucleic acids of active autotrophic nitrifiers. Analysis of 13C-associated 16S rRNA gene fragments, following CsCl density centrifugation, by cloning and DGGE indicated sequences highly similar to the AOB Nitrosomonas sp. Nm143 and Nitrosomonas cryotolerans and to the nitrite oxidizer Nitrospira marina. No sequence with similarity to the Nitrosospira cluster 1 clade was recovered during SIP analysis. The potential role of Nitrosospira cluster 1 in autotrophic ammonia oxidation therefore remains uncertain.

Ammonia↗
PubMed2001

Nitrification and denitrification as a source for NO and NO2 production in high-strength wastewater.

Laboratory and half-technical scale experiments were performed to evaluate nitric oxide (NO) and nitrogen dioxide (NO2) production during biological N-elimination from wastewater with high ammonium concentration (about 700 mg N L-1). In a laboratory scale bioreactor with biomass retention, the ammonia oxidizer Nitrosomonas europaea and the denitrifier Paracoccus denitrificans were grown as reference organisms in co-culture in order to simulate the nitrifying and denitrifying community of wastewater treatment plants. Synthetic wastewater and sludge liquor from the municipal wastewater treatment plant in Lueneburg (Germany) were used. In the laboratory scale reactor, during the treatment of synthetic wastewater, 0.28% of the oxidized ammonium-N was released as NO-N by a pure culture of Nitrosomonas. A simultaneously nitrifying and denitrifying co-culture only released 0.04 to 0.2%. NO2 formation was not observed. NO production was much higher in sludge liquor. A pure culture of Nitrosomonas produced 0.52% NO + NO2-N (= NOx-N), a co-culture of Nitrosomonas and Paracoccus even 1.64% NOx-N. The production rate strongly depended on the media and the organisms used. In a co-culture of N. europaea and P denitrificans, Nitrosomonas was shown to be the most efficient NO producer. NO production increased with ammonium oxidation rate and with nitrite concentration of the medium. In synthetic wastewater, NO production was not influenced by reduced oxygen content. However, in sludge liquor NO production rate increased with decreasing O2 concentration. Here, for the first time, the formation of significant amounts of NO2 during simultaneous nitrification/denitrification could be demonstrated. In half-technical scale experiments, only 0.07% of the oxidized ammonium-N was released as NO-N from the nitrification stage. NO2 was not detectable. Release of nitric oxide from the denitrification stage was mainly diffusion limited and the amount produced did not exceed 0.0001%. A calculation on the basis of the results presented, revealed that biological treatment of nitrogen-rich wastewater is not a significant source for pollution of the atmosphere with NOx in industrial areas.

Biodegradation, Environmental↗
PubMed2002

Growth at low ammonium concentrations and starvation response as potential factors involved in niche differentiation among ammonia-oxidizing bacteria.

In nature, ammonia-oxidizing bacteria have to compete with heterotrophic bacteria and plants for limiting amounts of ammonium. Previous laboratory experiments conducted with Nitrosomonas europaea suggested that ammonia-oxidizing bacteria are weak competitors for ammonium. To obtain a better insight into possible methods of niche differentiation among ammonia-oxidizing bacteria, we carried out a growth experiment at low ammonium concentrations with N. europaea and the ammonia oxidizer G5-7, a close relative of Nitrosomonas oligotropha belonging to Nitrosomonas cluster 6a, enriched from a freshwater sediment. Additionally, we compared the starvation behavior of the newly enriched ammonia oxidizer G5-7 to that of N. europaea. The growth experiment at low ammonium concentrations showed that strain G5-7 was able to outcompete N. europaea at growth-limiting substrate concentrations of about 10 micro M ammonium, suggesting better growth abilities of the ammonia oxidizer G5-7 at low ammonium concentrations. However, N. europaea displayed a more favorable starvation response. After 1 to 10 weeks of ammonium deprivation, N. europaea became almost immediately active after the addition of fresh ammonium and converted the added ammonium within 48 to 96 h. In contrast, the regeneration time of the ammonia oxidizer G5-7 increased with increasing starvation time. Taken together, these results provide insight into possible mechanisms of niche differentiation for the ammonia-oxidizing bacteria studied. The Nitrosomonas cluster 6a member, G5-7, is able to grow at ammonium concentrations at which the growth of N. europaea, belonging to Nitrosomonas cluster 7, has already ceased, providing an advantage in habitats with continuously low ammonium concentrations. On the other hand, the ability of N. europaea to become active again after longer periods of starvation for ammonium may allow better exploitation of irregular pulses of ammonium in the environment.

Ammonia↗