Amplification of 16S ribosomal RNA genes of autotrophic ammonia-oxidizing bacteria demonstrates the ubiquity of nitrosospiras in the environment.
(25/31)
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. (+info)
Nitrification in the intertidal zone: influence of effluent type and effect of tannin on nitrifiers.
(26/31)
Nitrification by intertidal sediments was measured by using a tide simulator that approximated the cycle of seawater on tidal flats. Sediments were chosen from sites affected by industrial and municipal effluents and pastoral seepage and runoff. The ability of sediments from different sites to nitrify endogenous nitrogen varied markedly. All sites exhibited an initial lag before activity commenced. The duration of this lag and the rate of nitrate production were different at each site. The sediments were also capable of oxidizing NH3-N supplied to them in seawater. This "nitrification potential" was highest at sites receiving nitrogenous effluents (slaughterhouse and sewage), but was also substantial in sediments affected by bark extract effluent and pasture runoff. The lowest potential and the longest lag were exhibited by sediments in an apple cannery effluent area. Enrichment cultures of nitrifying microorganisms were obtained from all sites using NH4+ as a source of energy, but enrichments for nitrite oxidizers were unsuccessful. Concentrated pine bark tannins, similar in origin to those in effluents at the well-nitrifying chipmill site, were tested for toxicity to pure cultures of nitrifying bacteria. Two Nitrobacter strains and one Nitrosomonas strain were unaffected by tannins even at 5 mg/ml. A Nitrosolobus and a Nitrosospira strain were inhibited partially at 5 mg/ml and only slightly or not at all at 1 mg/ml. (+info)
In situ morphology of nitrifying-like bacteria in aquaculture systems.
(27/31)
The in situ microbiota from several aquaculture facilities with active nitrification was examined by transmission electron microscopy of thin sections for the presence of bacteria that contained intracytoplasmic membranes characteristic of the nitrifying bacteria. Colonies of bacteria with the cellular morphology of a species of Nitrosomonas were found to be present in both the culture water and in the biological filter slime of a freshwater chinook salmon (Oncorhynchus tshawytscha) culture system. bacteria in the water possessed the normal nitrosomonas type of ultrastructure, whereas similar bacteria in the slime had an aberrant morphology due to multiple invaginations of the cell wall and cyto-membranes and a significantly greater number of ribosomes. These nitrosomonas-like bacteria lysed during enrichment in commonly used media. Bacteria with the morphology of species of Nitrosomonas and Nitrosococcus were also observed in colonies in the surface slimes of marine culture systems for striped bass (Morone saxatilis) and quahaug (Mercenaria mercenaria). (+info)
Combined molecular and conventional analyses of nitrifying bacterium diversity in activated sludge: Nitrosococcus mobilis and Nitrospira-like bacteria as dominant populations.
(28/31)
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. (+info)
Assessment of changes in microbial community structure during operation of an ammonia biofilter with molecular tools.
(29/31)
Biofiltration has been used for two decades to remove odors and various volatile organic and inorganic compounds in contaminated off-gas streams. Although biofiltration is widely practiced, there have been few studies of the bacteria responsible for the removal of air contaminants in biofilters. In this study, molecular techniques were used to identify bacteria in a laboratory-scale ammonia biofilter. Both 16S rRNA and ammonia monooxygenase (amoA) genes were used to characterize the heterotrophic and ammonia-oxidizing bacteria collected from the biofilter during a 102-day experiment. The overall diversity of the heterotrophic microbial population appeared to decrease by 38% at the end of the experiment. The community structure of the heterotrophic population also shifted from predominantly members of two subdivisions of the Proteobacteria (the beta and gamma subdivisions) to members of one subdivision (the gamma subdivision). An overall decrease in the diversity of ammonia monooxygenase genes was not observed. However, a shift from groups dominated by organisms containing Nitrosomonas-like and Nitrosospira-like amoA genes to groups dominated by organisms containing only Nitrosospira-like amoA genes was observed. In addition, a new amoA gene was discovered. This new gene is the first freshwater amoA gene that is closely affiliated with Nitrosococcus oceanus and the particulate methane monooxygenase gene from the methane oxidizers belonging to the gamma subdivision of the Proteobacteria. (+info)
Molecular analysis of ammonia-oxidizing bacteria of the beta subdivision of the class Proteobacteria in compost and composted materials.
(30/31)
Although the practice of composting animal wastes for use as biofertilizers has increased in recent years, little is known about the microorganisms responsible for the nitrogen transformations which occur in compost and during the composting process. Ammonia is the principle available nitrogenous compound in composting material, and the conversion of this compound to nitrite in the environment by chemolithotrophic ammonia-oxidizing bacteria is an essential step in nitrogen cycling. Therefore, the distribution of ammonia-oxidizing members of the beta subdivision of the class Proteobacteria in a variety of composting materials was assessed by amplifying 16S ribosomal DNA (rDNA) and 16S rRNA by PCR and reverse transcriptase PCR (RT-PCR), respectively. The PCR and RT-PCR products were separated by denaturing gradient gel electrophoresis (DGGE) and were identified by hybridization with a hierarchical set of oligonucleotide probes designed to detect ammonia oxidizer-like sequence clusters in the genera Nitrosospira and Nitrosomonas. Ammonia oxidizer-like 16S rDNA was detected in almost all of the materials tested, including industrial and experimental composts, manure, and commercial biofertilizers. A comparison of the DGGE and hybridization results after specific PCR and RT-PCR suggested that not all of the different ammonia oxidizer groups detected in compost are equally active. amoA, the gene encoding the active-site-containing subunit of ammonia monooxygenase, was also targeted by PCR, and template concentrations were estimated by competitive PCR. Detection of ammonia-oxidizing bacteria in the composts tested suggested that such materials may not be biologically inert with respect to nitrification and that the fate of nitrogen during composting and compost storage may be affected by the presence of these organisms. (+info)
Phylogenetic differences between particle-associated and planktonic ammonia-oxidizing bacteria of the beta subdivision of the class Proteobacteria in the Northwestern Mediterranean Sea.
(31/31)
The aim of this study was to determine if there were differences between the types of ammonia-oxidizing bacteria of the beta subdivision of the class Proteobacteria associated with particulate material and planktonic samples obtained from the northwestern Mediterranean Sea. A nested PCR procedure performed with ammonia oxidizer-selective primers was used to amplify 16S rRNA genes from extracted DNA. The results of partial and full-length sequence analyses of 16S rRNA genes suggested that different groups of ammonia-oxidizing bacteria were associated with the two sample types. The particle-associated sequences were predominantly related to Nitrosomonas eutropha, while the sequences obtained from the planktonic samples were related to a novel marine Nitrosospira group (cluster 1) for which there is no cultured representative yet. A number of oligonucleotide probes specific for different groups of ammonia oxidizers were used to estimate the relative abundance of sequence types in samples of clone libraries. The planktonic libraries contained lower proportions of ammonia oxidizer clones (0 to 26%) than the particulate material libraries (9 to 83%). Samples of the planktonic and particle-associated libraries showed that there were depth-related differences in the ammonia oxidizer populations, with the highest number of positive clones in the particle-associated sample occurring at a depth of 700 m. The greatest difference between planktonic and particle-associated populations occurred at a depth of 400 m, where only 4% of the clones in the planktonic library were identified as Nitrosomonas clones, while 96% of these clones were identified as clones that were related to the marine Nitrosospira species. Conversely, all ammonia oxidizer-positive clones obtained from the particle-associated library were members of the Nitrosomonas group. This is the first indication that Nitrosomonas species and Nitrosospira species may occupy at least two distinct environmental niches in marine environments. The occurrence of these groups in different niches may result from differences in physiological properties and, coupled with the different environmental conditions associated with these niches, may lead to significant differences in the nature and rates of nitrogen cycling in these environments. (+info)