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PubMed2004

Occurrence of nitrifiers and diversity of ammonia-oxidizing bacteria in developing drinking water biofilms.

We studied the population dynamics of nitrifying bacteria during the development of biofilms up to 233 or 280 days on polyvinylchloride pipes connected to two full-scale drinking water distribution networks supplying processed and chloraminated surface water. The numbers of nitrifiers in biofilms were enumerated at intervals of 10-64 days by the most probable number (MPN) method at waterworks and at several study sites in distribution network areas. The numbers of nitrifiers increased towards the distal sites. The highest detected MPN counts of ammonia-oxidizing bacteria (AOB) for study areas 1 and 7 were 500 MPN cm(-2) and 1.0 x 10(6) MPN cm(-2), and those of nitrite-oxidizing bacteria (NOB) 96 MPN cm(-2) and 2.2 x 10(3) MPN cm(-2), respectively. The diversity of AOB was determined by PCR amplifying, cloning and sequencing the partial ammonia monooxygenase (amoA) gene of selected biofilm samples presenting different biofilm ages. The PCR primers used, A189 and A682, also amplified a fragment of particulate methane monooxygenase (pmoA) gene of methane-oxidizing bacteria. The majority of biofilm clones (24 out of 30 studied) contained Nitrosomonas amoA-like sequences. There were only two pmoA-like sequences of Type I methanotrophs, and four sequences positioned in amoA/pmoA sequence groups of uncultured bacteria. From both study area very similar or even completely identical Nitrosomonas amoA-like sequences were obtained despite of high difference in AOB numbers. The results show that the conditions in newly formed biofilms in drinking water distribution systems favor the growth of Nitrosomonas-type AOB.

Ammonia↗

PubMed2002

Denaturing gradient gel electrophoresis (DGGE) approaches to study the diversity of ammonia-oxidizing bacteria.

Denaturing gradient gel electrophoresis (DGGE) of PCR amplicons of the ammonia monooxygenase gene (amoA) was developed and employed to investigate the diversity of ammonia-oxidizing bacteria (AOB) in four different habitats. The results were compared to DGGE of PCR-amplified partial 16S rDNA sequences made with primers specific for ammonia-oxidizing bacteria. Potential problems, such as primer degeneracy and multiple gene copies of the amoA gene, were investigated to evaluate and minimize their possible impact on the outcome of a DGGE analysis. amoA and 16S rDNA amplicons were cloned, and a number of clones screened by DGGE to determine the abundance of different motility types in the clone library. The abundance of clones was compared to the relative intensity of bands emerging in the band pattern produced by direct amplification of the genes from the environmental sample. Selected clones were sequenced to evaluate the specificity of the respective primers. The 16S rDNA primer pair, reported to be specific for ammonia-oxidizing bacteria (AOB), generated several sequences that were not related to the known Nitrosospira-Nitrosomonas group and, thus, not likely to be ammonia oxidizers. However, no false positives were found among the sequences retrieved with the modified amoA primers. Some phylogenetic information could be deduced from the position of amoA bands in DGGE gels. The Nitrosomonas-like sequences were found within a denaturant range from 30% to 46%, whereas the Nitrosospira-like sequences migrated to 50% to 60% denaturant. The majority of retrieved sequences from all four habitats with high ammonia loads were Nitrosomonas-like and only few Nitrosospira-like sequences were detected.

Ammonia↗

PubMed1968

Properties of some reductase enzymes in the nitrifying bacteria and their relationship to the oxidase systems.

The reductase enzymes in Nitrosomonas and Nitrobacter were studied under anaerobic conditions when the oxidase enzymes were inactive. The most effective electron-donor systems for nitrate reductase in Nitrobacter were reduced benzyl viologen alone, phenazine methosulphate with either NADH or NADPH, and FMN or FAD with NADH. Nitrite and hydroxylamine reductases were found in both nitrifying bacteria, and optimum activity for each enzyme was obtained with NADH or NADPH with either FMN or FAD. The product of both these enzymes was identified as ammonia. In extracts of Nitrosomonas the ammonia was further utilized by an NADPH-specific glutamate dehydrogenase. (15)N-labelled nitrite, hydroxylamine and ammonia were rapidly incorporated into cell protein by Nitrosomonas, and Nitrobacter in addition incorporated [(15)N]nitrate. Relatively gentle methods of cell disruption were compared with ultrasonic treatment, to enable a more exact study to be undertaken of the intracellular distribution of the oxidase and reductase enzymes. The functional relationship of these opposing enzyme systems in the nitrifying bacteria is considered.

Ammonia↗

PubMed2004

Nitrification and anammox with urea as the energy source.

Urea is present in many ecosystems and can be used as an energy source by chemolithotrophic aerobic ammonia oxidizing bacteria (AOB). Thus the utilization of urea in comparison to ammonia, by AOB as well as anaerobic ammonia oxidizing (Anammox) bacteria was investigated, using enrichments cultures, inoculated with activated sludge, and molecular ecological methods. In batch enrichment cultures grown with ammonia a population established in 2 weeks, which was dominated by halophilic and halotolerant AOB as determined by fluorescence in situ hybridization (FISH) experiments, with the 16S rRNA targeting oligonucleotide probe NEU. In other batch enrichment cultures using urea, the AOB population was assessed by PCR amplification, cloning and phylogenetic analysis of amoA and ribosomal 16S rRNA genes. While only one of the 48 16S rRNA gene clones could be identified as AOB (Nitrosomonas oligotropha), the amoA approach revealed two more AOB, Nitrosomonas europaea and Nitrosomonas nitrosa to be present in the enrichment. FISH analysis of the enrichment with probe NEU and newly designed probes for a specific detection of N. oligotropha and N. nitrosa related organisms, respectively, showed that N. oligotropha-like AOB formed about 50% of the total bacterial population. Also N. nitrosa (about 15% of the total population) and N. europaea (about 5% of the total population) were relatively abundant. Additionally, continuous enrichments were performed under oxygen limitation. When ammonia was the energy source, the community in this reactor consisted of Anammox bacteria and AOB hybridizing with probe NEU. As the substrate was changed to urea, AOB related to N. oligotropha became the dominant AOB in this oxygen limited consortium. This resulted in a direct conversion of urea to dinitrogen gas, without the addition of organic carbon.

Aerobiosis↗

PubMed2001

Factors affecting nitrite build-up in nitrifying biofilm reactor.

Nitrite build-up in a nitrifying biofilm reactor for direct denitrification from the nitrite stage was investigated. At least three factors were found to influence the nitrite build-up: (i) the relative specific growth rates of Nitrosomonas to Nitrobacter, microNs/microNb in the biofilm; (ii) the relative initial ratio between Nitrosomonas and Nitrobacter on the support surface, (Mao)Ns/(Mao)Nb; (iii) the level of free ammonia, particularly at greater than 0.1 mg N/l that can be inhibitory to Nitrobacter. Results showed that without free ammonia inhibition, the first two factors determined the degree of nitrite build-up. The effect of (Mao)Ns and (Mao)Nb on nitrification kinetics were more significant than the specific growth rates of Nitrosomonas and Nitrobacter. By regulating effects of these three factors, extremely high concentrations of nitrite build-up could occur in a biofilm reactor. This offers a potential for achieving direct denitrification through a nitrate shunt.

Ammonia↗

PubMed2005

Community survey of ammonia-oxidizing bacteria in full-scale activated sludge processes with different solids retention time.

AIMS: To study the effects of different solids retention time (SRT) on the nitrification activity and community composition of ammonia-oxidizing bacteria (AOB) in two full-scale activated sludge processes during a 5-month period. METHODS AND RESULTS: The AOB community composition was analysed using fluorescence in situ hybridization (FISH) and denaturing gradient gel electrophoresis (DGGE), and the identified populations were enumerated by quantitative FISH. Potential nitrification rates were determined in batch tests and the in situ rates were calculated from mass balances of nitrogen in the plants. Increased SRT reduced the nitrification activity, but neither the number per mixed liquor suspended solids nor community composition of AOB were affected. Two dominant AOB populations related to Nitrosomonas europaea and Nitrosomonas oligotropha were identified by FISH, whereas only the latter could be detected by DGGE. CONCLUSIONS: The effect of a longer SRT on the activity was probably because of physiological changes in the AOB community rather than a change in community composition. SIGNIFICANCE AND IMPACT OF THE STUDY: Physiological alterations of a stable AOB community are possible and may stabilize activated sludge processes. The commonly used FISH probes designed to target all beta-proteobacterial AOB does not detect certain Nitrosomonas oligotropha populations, leading to an underestimation of AOB if a wider set of probes is not used.

Ammonia↗

PubMed2006

Vertical distribution of nitrifying populations in bacterial biofilms from a full-scale nitrifying trickling filter.

Cryosectioned biofilm from three depths (0.5, 3.0 and 6.0 m) in a full-scale nitrifying trickling filter (NTF) were studied using fluorescence in situ hybridization (FISH). A large number of sections were used to determine how the biofilm thickness, structure and community composition varied with depth along the ammonium concentration gradient in the NTF, and how the ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) were distributed vertically within the biofilm. Both the biofilm thickness and relative biomass content of the biofilm decreased with depth, along with structural differences such as void size and surface roughness. Four AOB populations were found, with two Nitrosomonas oligotropha populations dominating at all depths. A smaller population of Nitrosomonas europaea was present only at 0.5 m, while a population of Nitrosomonas communis increased with depth. The two N. oligotropha populations showed different vertical distribution patterns within the biofilm, indicating different ecophysiologies even though they belong to the same AOB lineage. All NOB were identified as Nitrospira sp., and were generally more associated with the biofilm base than the surface-associated dominating AOB population. Additionally, a small population of anaerobic ammonia-oxidizers was found at 6.0 m, even though the biofilm was well aerated.

Ammonia↗

PubMed2006

Microbial community development in a laboratory-scale nitrifying activated sludge system with input from a side-stream bioreactor treating digester supernatant.

Three laboratory-scale activated sludge treatment trains were operated to investigate the effect on biodiversity in plug flow (PFR) main-stream sewage treatment from input of biomass from side-stream reactors treating anaerobic digester supernatant. One train had a completely mixed (CSTR) side-stream reactor, one a PFR side-stream reactor, and the third train was a control that did not receive input from a side-stream reactor. Restriction endonucleases were used to digest polymerase chain reaction-amplified ammonia monooxygenase subunit A (amoA) genes in monthly samples from each reactor. Restriction fragment banding patterns from polyacrylimide gel electrophoresis indicated that the structure of the ammonia oxidizing bacteria (AOB) populations in all five reactors stabilized by the fourth month of operation and then did not vary subsequently. Furthermore, a dendrogram generated using the Jaccard distance showed that the AOB in each side-stream reactor was most similar to the main-stream reactor in the same train indicating that the AOB population in the side-stream reactor exerts a strong influence on the population in the main-stream reactor. Sequencing results indicated that Nitrosomonas europea, an r-strategist, was the dominant AOB in the PFR side-stream reactor, while Nitrosomonas europea and Nitrosomonas marina, a marine bacterium, were strongly represented in the CSTR side-stream reactor.

Base Sequence↗

PubMed2006

Nitrogen removal rates at a technical-scale pilot plant with the one-stage partial nitritation/Anammox process.

Traditional nitrification/denitrification is not suitable for nitrogen removal when wastewater contains high concentrations of ammonium nitrogen and low concentrations of biodegradable carbon. Recently, a deammonification process was developed and proposed as a new technology for treatment of such streams. This process relies on a stable interaction between aerobic bacteria Nitrosomonas, that accomplish partial nitritation and anaerobic bacteria Planctomycetales, which conduct the Anammox reaction. Simultaneous performance of these two processes can lead to a complete autotrophic nitrogen removal in one single reactor. The experiments where nitrogen was removed in one reactor were performed at a technical-scale moving-bed pilot plant, filled with Kaldnes rings and supplied with supernatant after dewatering of digested sludge. It was found that a nitrogen removal rate obtained at the pilot plant was 1.9 g m(-2) d(-1). Parallel to the pilot plant run, a series of batch tests were carried out under anoxic and aerobic conditions. Within the batch tests, where the pilot plant's conditions were simulated, removal rates reached up to 3 g N m(-2)d(-1). Moreover, the batch tests with inhibition of Nitrosomonas showed that only the Anammox bacteria (not anoxic removal by Nitrosomonas) are responsible for nitrogen removal.

Aerobiosis↗

PubMed2001

Energy model and metabolic flux analysis for autotrophic nitrifiers.

The behavior of pure cultures of nitrifying microorganisms under autotrophic growth operating conditions was investigated and the relations between their energy metabolism and their anabolism analyzed by means of metabolic network computation. The description of the metabolism of the nitrifiers is extended to their energy metabolism by introducing compartmentalization (cytoplasmic and periplasmic sides) and studying coupling between the electron transport chain and the proton gradient generation. The energy model of Nitrosomonas and Nitrobacter was developed based on the oxidoreduction reactions known to be involved. The electron transport chains and the associated proton translocation for these models are described. Several possible hypotheses are analyzed and discussed concerning the thermodynamic consistency of all the oxidoreduction reactions. For Nitrosomonas, the most delicate point is the second step of hydroxylamine oxidation. For Nitrobacter a new energy model is proposed in which NO plays an important role as node in the distribution of electrons from NO(2)(-) oxidation to the membrane electron transport chain. The compartmentalization enables us to consider a proton gradient dissipation flux as the expression of the overall energy loss in metabolic analysis (the so-called maintenance phenomena). The energy model (electron transport chain, proton gradient) is associated with an overall description of the metabolism of Nitrosomonas and Nitrobacter in terms of metabolic flux calculation. This representation demonstrates that a maintenance in nitrifiers expressed as a proton leak is no higher than for other aerobes. The yields calculated from the energy models integrated with the metabolic models of nitrifiers are consistent with the experimental yields in the literature.

Ammonia↗

PubMed1998

Functional Gene Hybridization Patterns of Terrestrial Ammonia-Oxidizing Bacteria.

Abstract The biochemical pathway and genetics of autotrophic ammonia oxidation have been studied almost exclusively in Nitrosomonas europaea. Terrestrial autotrophic ammonia-oxidizing bacteria (AAOs), however, comprise two distinct phylogenetic groups in the beta-Proteobacteria, the Nitrosomonas and Nitrosospira groups. Hybridization patterns were used to assess the potential of functional probes in non-PCR-based molecular analysis of natural AAO populations and their activity. The objective of this study was to obtain an overview of functional gene homologies by hybridizing probes derived from N. europaea gene sequences ranging in size from 0.45 to 4.5 kb, and labeled with 32P to Southern blots containing genomic DNA from four Nitrosospira representatives. Probes were specific for genes encoding ammonia monooxygenase (amoA and amoB), hydroxylamine oxidoreductase (hao), and cytochrome c-554 (hcy). These probes produced hybridization signals, at low stringency (30 degreesC), with DNA from each of the four representatives; signals at higher stringency (42 degreesC) were greatly reduced or absent. The hybridization signals at low stringency ranged from 20 to 76% of the total signal obtained with N. europaea DNA. These results indicate that all four functional genes in the ammonia oxidation pathway have diverged between the Nitrosomonas and Nitrosospira groups. The hao probe produced the most consistent hybridization intensities among the Nitrosospira representatives, suggesting that hao sequences would provide the best probes for non-PCR-based molecular analysis of terrestrial AAOs. Since N. europaea can also denitrify, an additional objective was to hybridize genomic DNA from AAOs with probes for Pseudomonas genes involved in denitrification. These probes were specific for genes encoding heme-type dissimilatory nitrite reductase (dNir), Cu-type dNir, and nitrous oxide reductase (nosz). No hybridization signals were observed from probes for the heme-type dNir or nosz, but Nitrosospira sp. NpAV and Nitrosolobus sp. 24-C hybridized, under low-stringency conditions, with the Cu-type dNir probe. These results indicate that AAOs may also differ in their mechanisms and capacities for denitrification.

Journal Article↗

PubMed2006

Development and application of real-time PCR for quantification of specific ammonia-oxidizing bacteria in activated sludge of sewage treatment systems.

In this study, four real-time polymerase chain reaction (PCR) primer sets were developed for the 16S rRNA genes of specific ammonia-oxidizing bacteria (AOB) found in activated sludge of sewage treatment systems. The primer sets target two of several sequence types of the Nitrosomonas oligotropha cluster, members within the Nitrosomonas communis cluster, and all members of the Nitrosomonas europaea-Nitrosococcus mobilis cluster. The detection limit of each primer set was in the range of 3x10(1)-6x10(2) genes reaction(-1). Reliable quantification of the target AOB DNA was obtained when the target AOB DNA comprised more than 0.1% of total AOB DNA in the sample. The application of the primer sets to samples taken from five sewage treatment systems showed that, in all systems, the majority of the AOB population was comprised of one sequence type of the N. oligotropha cluster (3.9+/-1.5x10(9)-1.7+/-0.5x10(10) cell l(-1)) and, in most systems, followed by members within the N. communis cluster (2.8+/-0.3x10(9)-1.0+/-0.1x10(10) cell l(-1)) or/and another sequence type of the N. oligotropha cluster (1.5+/-0.6x10(8)-5.5+/-0.5x10(8) cell l(-1)). N. europaea-N. mobilis cluster arose solely in small numbers (4.9+/-0.8x10(8) cell l(-1)) in one system. Real-time PCR-amplified products obtained from genomic DNA extracted from samples were verified using clone library, and it revealed that only the target AOB DNA were PCR amplified, without amplification of the nontarget sequences.

Ammonia↗

PubMed2002

Detection of Proteobacteria from the rumen by PCR using methanotroph-specific primers.

AIMS: To detect Proteobacteria, including methanotrophs, from the rumen fluid and the bacteria inhabiting the rumen epithelium. METHODS AND RESULTS: Proteobacteria inhabiting the rumen were detected by PCR using methanotroph-specific primers. The detected Proteobacteria were divided into clusters A, B, and C in addition to one clone, which was distinct from the clusters and closely related to Nitrosomonas sp. The clusters A, B, and C were close to Succinivibrio dextrinosolvens, Enterobacter cloacae, and Actinobacillus minor, respectively. The clones obtained from the rumen fluid each belonged to cluster A or B. The clones obtained from the rumen epithelium belonged to cluster B or C or to Nitrosomonas sp. CONCLUSIONS: It has been assumed that the rumen fluid and the rumen epithelium host different populations of Proteobacteria. Moreover, detection of Nitrosomonas from the rumen epithelium would indicate the possibility that the bacterium oxidizes ammonia and methane on the rumen surface. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings suggest that the rumen fluid and the epithelium support different microbial populations, which would play specific roles in rumen function. Future study should focus on the relationship between these communities and physiological functions in the rumen.

Animals↗

PubMed2006

Nitrosospira spp. can produce nitrous oxide via a nitrifier denitrification pathway.

Nitrous oxide (N(2)O) emission from soils is a major contributor to the atmospheric loading of this potent greenhouse gas. It is thought that autotrophic ammonia oxidizing bacteria (AOB) are a significant source of soil-derived N(2)O and a denitrification pathway (i.e. reduction of NO(2) (-) to NO and N(2)O), so-called nitrifier denitrification, has been demonstrated as a N(2)O production mechanism in Nitrosomonas europaea. It is thought that Nitrosospira spp. are the dominant AOB in soil, but little information is available on their ability to produce N(2)O or on the existence of a nitrifier denitrification pathway in this lineage. This study aims to characterize N(2)O production and nitrifier denitrification in seven strains of AOB representative of clusters 0, 2 and 3 in the cultured Nitrosospira lineage. Nitrosomonas europaea ATCC 19718 and ATCC 25978 were analysed for comparison. The aerobically incubated test strains produced significant (P < 0.001) amounts of N(2)O and total N(2)O production rates ranged from 2.0 amol cell(-1) h(-1), in Nitrosospira tenuis strain NV12, to 58.0 amol cell(-1) h(-1), in N. europaea ATCC 19718. Nitrosomonas europaea ATCC 19718 was atypical in that it produced four times more N(2)O than the next highest producing strain. All AOB tested were able to carry out nitrifier denitrification under aerobic conditions, as determined by production of (15)N-N(2)O from applied (15)N-NO(2) (-). Up to 13.5% of the N(2)O produced was derived from the exogenously applied (15)N-NO(2) (-). The results suggest that nitrifier denitrification could be a universal trait in the betaproteobacterial AOB and its potential ecological significance is discussed.

Aerobiosis↗

PubMed2006

Molecular analysis of ammonia-oxidizing bacteria community in intermittent aeration sequencing batch reactors used for animal wastewater treatment.

Bacterial communities and betaproteobacterial ammonia-oxidizing bacteria (AOB) communities were evaluated seasonally in an intermittent-aeration sequencing batch process (SBR, plant A) and in 12 other livestock wastewater treatment plants (WWTP): eight SBRs and four conventional activated-sludge systems. Microbial communities were analysed by reverse transcription polymerase chain reaction followed by denaturing-gradient gel electrophoresis (DGGE) and the construction of clone libraries for 16S rRNA and ammonia monooxygenase (amoA) genes. In plant A, the dominant bacteria were as-yet-uncultured bacteria of Bacteroidetes and Proteobacteria, and the DGGE profiles showed that the bacterial communities were stable during a given treatment cycle, but changed seasonally. In betaproteobacterial AOB communities, two AOB phylotypes (members of the Nitrosomonas ureae-oligotropha-marina cluster) were dominant during the seasons in plant A. Although the dominant AOB phylotypes differed among the 13 WWTPs, dominance by one or two AOB phylotypes was commonly observed in all plants. Sequencing of the DGGE bands indicated that amoA sequences belonging to the Nitrosomonas europaea-eutropha cluster were dominant in 11 plants, where the ammonia-nitrogen concentration was high in the raw wastewater, whereas those belonging to the Nitrosomonas ureae-oligotropha-marina cluster were dominant in two plants where the concentration was relatively low. Even though we detected many minor amoA sequences by means of five clone libraries for the A to D plants, no libraries comprised both amoA sequences belonging to the two clusters, indicating that the dominant AOBs were defined by cluster level in each plant.

Ammonia↗

PubMed1998

Application of molecular biological techniques to a seasonal study of ammonia oxidation in a eutrophic freshwater lake

The autotrophic ammonia-oxidizing bacteria in a eutrophic freshwater lake were studied over a 12-month period. Numbers of ammonia oxidisers in the lakewater were small throughout the year, and tangential-flow concentration was required to obtain meaningful estimates of most probable numbers. Sediments from littoral and profundal sites supported comparatively large populations of these bacteria, and the nitrification potential was high, particularly in summer samples from the littoral sediment surface. In enrichment cultures, lakewater samples nitrified at low (0.67 mM) ammonium concentrations only whereas sediment samples exhibited nitrification at high (12.5 mM) ammonium concentrations also. Enrichments at low ammonium concentration did not nitrify when inoculated into high-ammonium medium, but the converse was not true. This suggests that the water column contains a population of ammonia oxidizers that is sensitive to high ammonium concentrations. The observation of nitrification at high ammonium concentration by isolates from some winter lakewater samples, identified as nitrosospiras by 16S rRNA probing, is consistent with the hypothesis that sediment ammonia oxidizers enter the water column at overturn. With only one exception, nested PCR amplification enabled the detection of Nitrosospira 16S rDNA in all samples, but Nitrosomonas (N. europaea-eutropha lineage) 16S rDNA was never obtained. However, the latter were part of the sediment and water column communities, because their 16S rRNA could be detected by specific oligonucleotide probing of enrichment cultures. Furthermore, a specific PCR amplification regime for the Nitrosomonas europaea ammonia monooxygenase gene (amoA) yielded positive results when applied directly to sediment and lakewater samples. Patterns of Nitrosospira and Nitrosomonas detection by 16S rRNA oligonucleotide probing of sediment enrichment cultures were complex, but lakewater enrichments at low ammonium concentration were positive for nitrosomonads and not nitrosospiras. Analysis of enrichment cultures has therefore provided evidence for the existence of subpopulations within the lake ammonia-oxidizing community distinguishable on the basis of ammonium tolerance and possibly showing a seasonal distribution between the sediment and water column.

Journal Article↗

PubMed1998

Analysis of beta-subgroup proteobacterial ammonia oxidizer populations in soil by denaturing gradient gel electrophoresis analysis and hierarchical phylogenetic probing

A combination of denaturing gradient gel electrophoresis (DGGE) and oligonucleotide probing was used to investigate the influence of soil pH on the compositions of natural populations of autotrophic beta-subgroup proteobacterial ammonia oxidizers. PCR primers specific to this group were used to amplify 16S ribosomal DNA (rDNA) from soils maintained for 36 years at a range of pH values, and PCR products were analyzed by DGGE. Genus- and cluster-specific probes were designed to bind to sequences within the region amplified by these primers. A sequence specific to all beta-subgroup ammonia oxidizers could not be identified, but probes specific for Nitrosospira clusters 1 to 4 and Nitrosomonas clusters 6 and 7 (J. R. Stephen, A. E. McCaig, Z. Smith, J. I. Prosser, and T. M. Embley, Appl. Environ. Microbiol. 62:4147-4154, 1996) were designed. Elution profiles of probes against target sequences and closely related nontarget sequences indicated a requirement for high-stringency hybridization conditions to distinguish between different clusters. DGGE banding patterns suggested the presence of Nitrosomonas cluster 6a and Nitrosospira clusters 2, 3, and 4 in all soil plots, but results were ambiguous because of overlapping banding patterns. Unambiguous band identification of the same clusters was achieved by combined DGGE and probing of blots with the cluster-specific radiolabelled probes. The relative intensities of hybridization signals provided information on the apparent selection of different Nitrosospira genotypes in samples of soil of different pHs. The signal from the Nitrosospira cluster 3 probe decreased significantly, relative to an internal control probe, with decreasing soil pH in the range of 6.6 to 3.9, while Nitrosospira cluster 2 hybridization signals increased with increasing soil acidity. Signals from Nitrosospira cluster 4 were greatest at pH 5.5, decreasing at lower and higher values, while Nitrosomonas cluster 6a signals did not vary significantly with pH. These findings are in agreement with a previous molecular study (J. R. Stephen, A. E. McCaig, Z. Smith, J. I. Prosser, and T. M. Embley, Appl. Environ. Microbiol 62:4147-4154, 1996) of the same sites, which demonstrated the presence of the same four clusters of ammonia oxidizers and indicated that selection might be occurring for clusters 2 and 3 at acid and neutral pHs, respectively. The two studies used different sets of PCR primers for amplification of 16S rDNA sequences from soil, and the similar findings suggest that PCR bias was unlikely to be a significant factor. The present study demonstrates the value of DGGE and probing for rapid analysis of natural soil communities of beta-subgroup proteobacterial ammonia oxidizers, indicates significant pH-associated differences in Nitrosospira populations, and suggests that Nitrosospira cluster 2 may be of significance for ammonia-oxidizing activity in acid soils.

Journal Article↗

PubMed2002

Ammonia- and nitrite-oxidizing bacterial communities in a pilot-scale chloraminated drinking water distribution system.

Nitrification in drinking water distribution systems is a common operational problem for many utilities that use chloramines for secondary disinfection. The diversity of ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) in the distribution systems of a pilot-scale chloraminated drinking water treatment system was characterized using terminal restriction fragment length polymorphism (T-RFLP) analysis and 16S rRNA gene (ribosomal DNA [rDNA]) cloning and sequencing. For ammonia oxidizers, 16S rDNA-targeted T-RFLP indicated the presence of Nitrosomonas in each of the distribution systems, with a considerably smaller peak attributable to Nitrosospira-like AOB. Sequences of AOB amplification products aligned within the Nitrosomonas oligotropha cluster and were closely related to N. oligotropha and Nitrosomonas ureae. The nitrite-oxidizing communities were comprised primarily of Nitrospira, although Nitrobacter was detected in some samples. These results suggest a possible selection of AOB related to N. oligotropha and N. ureae in chloraminated systems and demonstrate the presence of NOB, indicating a biological mechanism for nitrite loss that contributes to a reduction in nitrite-associated chloramine decay.

Ammonia↗