Diversity Of Ammonia Oxidizers Research Articles

Ammonia-oxidizing archaea and comammox Nitrospira clade B as freeze–thaw resistant nitrifiers in wetland soils

Wetland ecosystems are hotspots for nitrogen cycling, while the community dynamics of nitrification-driving microbes include ammonia-oxidizing archaea (AOA), ammonia-oxidizing bacteria (AOB), complete ammonia oxidizer (comammox) Nitrospira, and nitrite-oxidizing bacteria were not well-studied. In this study, we investigated the nitrification process in typical inland and coastal wetlands in northern China and found a high diversity of ammonia oxidizers. To simulate the freezing-thawing state under natural environment, the soil samples were first frozen at −20 °C for two months, and then gradually thawed at 4 °C. Further microcosm incubation combined with 13C-DNA-stable isotope probing analyses showed that after the freeze–thaw cycle, only the activities of AOA and comammox Nitrospira clade B recovered. The results indicated that these two groups of nitrifiers were more freeze–thaw resistant, which is in line with literature showing that AOA and comammox Nitrospira clade B were relatively more abundant than their counterparts in low-temperature environments. We tested, for the first time, the cold-shock resilience of nitrifying microbes in wetland soils and spotted candidates driving nitrification under fluctuating temperatures.

Ammonia oxidizers in the sea-surface microlayer of a coastal marine inlet.

Planktonic archaea are thought to play an important role in ammonia oxidation in marine environments. Data on the distribution, abundance, and diversity of ammonia oxidizers in the coastal sea-surface microlayer (SML) are lacking, despite previous reports of high abundance of Thaumarchaeota in the SML of estuaries and freshwater lakes. Here, we failed to detect the presence of ammonia-oxidizing bacteria in any of our samples taken from a semi-enclosed marine inlet in Japan. Therefore, we shifted our focus to examine the archaeal community composition as well as the Thaumarchaeota marine group I (MG-I) and ammonia monooxygenase subunit A (amoA) gene copy numbers and composition in the SML and corresponding underlying water (UW, 20 cm). amoA gene copy numbers obtained by quantitative PCR were consistent with the typical values observed in the surface waters of oceanic and coastal environments where nitrification activity has been detected, but the copy numbers were two- to three-fold less than those reported from the surface layers and UW of high mountain lakes. Both amoA and MG-I 16S rRNA gene copy numbers were significantly negatively correlated with chlorophyll-a and transparent exopolymer particle concentrations in the SML. Communities of archaea and ammonia-oxidizing archaea in SML samples collected during low wind conditions (≤5 m s–1) differed the most from those in UW samples, whereas the communities in SML samples collected during high wind conditions were similar to the UW communities. In the SML, low ratios of amoA to MG-I 16S rRNA genes were observed, implying that most of the SML Thaumarchaeota lacked amoA. To our knowledge, our results provide the first comparison of ammonia-oxidizing communities in the coastal SML with those in the UW.

Ammonia-oxidizing archaea are more sensitive than ammonia-oxidizing bacteria to long-term application of green manure in red paddy soil

Growing winter green manure is an effective method for improving nitrogen (N) management in paddy fields to enhance rice production. Ammonia oxidization is a key process in N cycling in these soils, but effects of green manuring on ammonia oxidizers have not been fully addressed for red paddy soils. The objective of this study was to investigate long-term impacts of winter green manure on abundance and diversity of ammonia oxidizers in rice paddy soils of Southern China. The field experiment established in 1982 included four treatments: rice-rice-winter fallow (RRF), rice-rice-ryegrass (RRG), rice–rice–rape (RRP) and rice–rice–milk vetch (RRV). The abundance and diversity of amoA genes from ammonia-oxidizing archaea (AOA) and bacteria (AOB) were quantified using quantitative PCR and 454 pyrosequencing, respectively. The AOA were more abundant than AOB in red paddy soils, with ratios of AOA to AOB from 36 to 1686. Long-term application of milk vetch increased the abundance of both AOA and AOB after green manures were incorporated. Some of the relative abundances of most abundant operational taxonomic units (OTUs) of AOA increased and others decreased after application of green manures, while most abundant OTUs of AOB remained unaffected. Redundancy analysis (RDA) found a clear separation between milk vetch and winter fallow, indicating that the community structure of AOA was influenced by application of milk vetch. Phylogenetic analysis showed that the most dominant OTUs of AOA and AOB were affiliated with Nitrososphaera and Nitrosospira, respectively. In conclusion, in red paddy soil, long-term application of milk vetch increased the abundances of AOA and AOB after incorporated. Moreover, long-term application of green manures had more profound influences on AOA community than on AOB in red paddy soils.

Responses of soil microbial community to nitrogen fertilizer and precipitation regimes in a semi-arid steppe

Changes of nitrogen (N) cycle caused by N fertilization and precipitation regimes have affected the key ecosystem structure and functions in temperate steppe, which may modify the structure of soil microbial communities involved in N transformation. This paper was designated to examine the response of soil ammonia oxidizers and denitrifiers to the N fertilization and precipitation regimes in a semi-arid steppe where N and water contents are major limiting factors of the grassland productivity. This study was based on a long-term N fertilization and precipitation regimes experiment in Inner Mongolia (116° 17′ 20″ E, 42° 2′ 29″ N). The treatments including CK (control), R (reduced precipitation), W (30% increase in precipitation), N (10 g N m−2 y−1), RN (reduced precipitation and 10 g N m−2 y−1), and WN (30% increase in precipitation and 10 g N m−2 y−1). Soil basic chemical properties and microbial activities were analyzed. Molecular methods were applied to determine the abundance, structure and diversity of ammonia oxidizers and denitrifiers. Statistical analysis detected the main and interactive effect of treatments on soil microbial communities and revealed the relationship between soil microbial community structures and environmental factors. N fertilization significantly increased ammonia-oxidizing bacteria (AOB) abundance. Ammonia-oxidizing archaea (AOA) community structure was markedly changed in N fertilizer treatment and strongly affected by soil pH, while soil nitrate and water content correlated with AOB community structure. Soil nitrate was the key factor influencing nirK gene community structure, while soil pH and water content explained much of the variations of nosZ gene community. AOB-amoA and nosZ gene community diversities were influenced by precipitation regimes and interaction of N fertilization and precipitation regimes, respectively. N fertilization and precipitation regimes had significant influences on the changes of soil properties and microbial functional communities. Soil nitrification was mainly driven by AOB in the semi-arid grassland. Changes of substrate content and soil pH were the key factors in shifting functional microbial communities. The non-synergistic effects of N fertilization and precipitation regimes on the microbial functional groups indicated that the negative effect of lower pH induced by N fertilization would be alleviated by precipitation regimes, which should be well considered in grassland restoration.

Effects of copper on the abundance and diversity of ammonia oxidizers during dairy cattle manure composting

This study investigated the effects of adding Cu(II) at two exposure levels (50 and 500mgkg−1, i.e., Cu50 and Cu500 treatments, respectively) on the activity of ammonia-oxidizing microorganisms during dairy cattle manure composting. The results showed that the pH, NH4+-N, NO3−-N, and potential ammonia oxidation values were inhibited significantly by the addition of Cu(II). Furthermore, the abundances of the ammonia-oxidizing archaea (AOA) amoA gene and ammonia-oxidizing bacteria (AOB) amoA gene were determined by quantitative PCR, and their compositions were evaluated by denaturing gradient gel electrophoresis (DGGE). AOA was the dominant ammonia oxidizing microorganism, of which the abundance was much higher than AOB during composting. Cu50 and Cu500 had significant inhibitory effects on the abundance of the amoA gene. The DGGE profile and statistical analysis showed that Cu(II) changed the AOA and AOB community structure and diversity, where Nitrosomonas and Crenarchaeota dominated throughout the composting process.

Long-term fertilization effects on active ammonia oxidizers in an acidic upland soil in China

The effects of long-term fertilization of acidic soils on ammonia-oxidizing archaea (AOA) and bacteria (AOB) communities and its ecological implications remain poorly understood. We chose an acidic upland soil site under long-term (27-year) fertilization to investigate ammonia oxidizer communities under four different regimes: mineral N fertilizer (N), mineral NPK fertilizer (NPK), organic manure (OM) and an unfertilized control (CK). Soil net nitrification rates were significantly higher in OM soils than in CK, N or NPK soils. Quantitative analysis of the distribution of amoA genes by DNA-based stable isotope probing revealed that AOA dominate in CK, N and NPK soils, while AOB dominate in OM soils. Denaturing gradient gel electrophoresis and clone library analyses of amoA genes revealed that Group 1.1a-associated AOA (also referred to as Nitrosotalea) were the most dominant active AOA population (>92%), while Nitrosospira Cluster 3 and Cluster 9 were predominant among active AOB communities. The functional diversity of active ammonia oxidizers in acidic soils is affected by long-term fertilization practices, and the responses of active ammonia oxidizers to mineral fertilizer and organic manure are clearly different. Our results provide strong evidence that AOA are more highly adapted to growth at low pH and low substrate availability than AOB, and they suggest that the niche differentiation and metabolic diversity of ammonia oxidizers in acidic soils are more complex than previously thought.

English

The assembling of microbial consortia in wastewater treatment facilities is a significance of environmental conditions. In the present research work, activated sludge from different wastewater treatment plants (WWTPs) were exploited at a molecular level to determine the influence of the complexity of the influent composition on the species structure and the diversity of bacterial consortia. The community fingerprints and technological data were subjected to the canonical correspondence and correlation analyses. The number of separated biological processes realized in the treatment line and the presence of industrial wastewater in the influent were the key factors determining the species structure of total and ammonia-oxidizing bacteria in biomass. The N2O-reducers community composition depended significantly on the design of the facility; the highest species richness of denitrifiers was noted in the WWTPs with separated denitrification tanks. The contribution of industrial streams to the inflow affected the diversity of total and denitrifying bacterial consortia and diminished the diversity of ammonia oxidizers. The obtained data are valuable for engineers since they revealed the main factors, including the design of wastewater treatment plant, influencing the microbial groups critical for the stability of purification processes.   Key words: Denitrification, waste water treatment plant, bacteria, influent. &nbsp

The influence of land use on the abundance and diversity of ammonia oxidizers.

Nitrification plays a significant role in soil nitrogen cycling, a process in which the first step can be catalyzed by ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB). In this study, six soil samples with distinct land-use regimes (forestland soil, paddy soil, wheat-planted soil, fruit-planted soil, grassland soil, and rape-planted soil) were collected from Chuzhou city in the Anhui province to elucidate the effects of land use on the abundance and diversity of AOA and AOB. The abundance of the archaeal amoA gene ranged from 2.12 × 10(4) copies per gram of dry soil to 2.57 × 10(5) copies per gram of dry soil, while the abundance of the bacterial amoA gene ranged from 5.58 × 10(4) copies per gram of dry soil to 1.59 × 10(8) copies per gram of dry soil. The grassland and the rape-planted soil samples maintained the highest abundance of the bacterial and archaeal amoA genes, respectively. The abundance of the archaeal amoA gene was positively correlated with the pH (P < 0.05). The ammonia concentrations exhibited a significantly positive relation with the abundance of the bacterial amoA gene (P < 0.01) and the number of OTUs of AOB (P < 0.05). The community composition of AOB was more sensitive to the land-use regimes than that of AOA. The data obtained in this study may be useful to better understand the nitrification process in soils with different land-use regimes.

Realization of Influent Waste Water on Microbial Community Structure of Activated Sludge Process

The assembling of microbial consortia in wastewater treatment facilities is a significance of environmental conditions. In the present research work, activated sludge from different wastewater treatment plants (WWTPs) were exploited at a molecular level to determine the influence of the complexity of the influent composition on the species structure and the diversity of bacterial consortia. The community fingerprints and technological data were subjected to the canonical correspondence and correlation analyses. The number of separated biological processes realized in the treatment line and the presence of industrial wastewater in the influent were the key factors determining the species structure of total and ammonia-oxidizing bacteria in biomass. The N<SUB>2</SUB>O-reducers community composition depended significantly on the design of the facility; the highest species richness of denitrifiers was noted in the WWTPs with separated denitrification tanks. The contribution of industrial streams to the inflow affected the diversity of total and denitrifying bacterial consortia and diminished the diversity of ammonia oxidizers. The obtained data are valuable for engineers since they revealed the main factors, including the design of wastewater treatment plant, influencing the microbial groups critical for the stability of purification processes.

Spatial and temporal diversity and abundance of ammonia oxidizers in semi-arid and arid soils: indications for a differential seasonal effect on archaeal and bacterial ammonia oxidizers

Besides water, nitrogen is the limiting factor for biomass production in arid ecosystems. Global climatic changes are exacerbating aridity levels, and the response of nitrogen-transforming microorganisms to these changes is not clear yet. Using semi-arid and arid ecosystems as surrogates for conditions of increased aridity, we investigated the activity, abundance, and diversity of ammonia-oxidizing bacteria (AOB) and archaea (AOA) in arid and semi-arid soils. Ammonia oxidation potentials were higher during the winter in both sites than in the summer, and higher nitrate concentrations were measured in the arid soil than in the semi-arid soil. Denaturing gradient gel electrophoresis (DGGE) patterns of AOB 16S rRNA gene fragments were similar for the arid and semi-arid soils with no seasonal variations. In contrast, the DGGE patterns of the AOA amoA gene fragments differed between the sites and a soil transfer experiment suggested that these differences are possibly associated with soil type. AOB numbers were higher during the winter than in the summer, while AOA numbers were higher during the summer. The results indicate the resistance of AOB and AOA community structure to arid conditions, albeit with seasonal variations in their abundance. Together, the results suggest the resilience of nitrification activity to increased aridity level.

Configuration of biological wastewater treatment line and influent composition as the main factors driving bacterial community structure of activated sludge

The structure of microbial consortia in wastewater treatment facilities is a resultant of environmental conditions created by the operational parameters of the purification process. In the research, activated sludge from nine Polish wastewater treatment plants (WWTPs) was investigated at a molecular level to determine the impact of the complexity of biological treatment line and the influent composition on the species structure and the diversity of bacterial consortia. The community fingerprints and technological data were subjected to the canonical correspondence and correlation analyses. The number of separated biological processes realized in the treatment line and the presence of industrial wastewater in the influent were the key factors determining the species structure of total and ammonia-oxidizing bacteria in biomass. The N2O-reducers community composition depended significantly on the design of the facility; the highest species richness of denitrifiers was noted in the WWTPs with separated denitrification tanks. The contribution of industrial streams to the inflow affected the diversity of total and denitrifying bacterial consortia and diminished the diversity of ammonia oxidizers. The obtained data are valuable for engineers since they revealed the main factors, including the design of wastewater treatment plant, influencing the microbial groups critical for the stability of purification processes.Electronic supplementary materialThe online version of this article (doi:10.1007/s11274-013-1273-9) contains supplementary material, which is available to authorized users.

Efficient ammonia removal from wastewater by a microbial biofilm in tuff-based intermittent biofilters

The aim of this study was to investigate the potential use of tuff as a matrix for ammonia removal in an intermittent flow biofilter (IBF) compared to sand. Sand and tuff filters were characterized regarding their respective total biofilm microbial biomass, numbers and diversity of ammonia oxidizers and the efficiency of ammonia removal. Microbial biomass per gram tuff was significantly higher than that of sand. However, ammonia was immediately removed using tuff filters, while its removal by sand filters started only after ten days, although the numbers of ammonia oxidizing bacteria (AOB) were higher in the sand filter. In addition, the tuff and sand filters were dominated by different species of AOB. The tuff filter was dominated by Nitrosospira species, while the sand filter was dominated by Nitrosomonas. In the practical level, tuff can be used as an efficient matrix in IBF for the removal of ammonia, possibly by creating a more suitable environment for bacterial activity than sand. This study suggests an available and cheap matrix to produce high quality effluents by IBF.

Niche specialization of terrestrial archaeal ammonia oxidizers

Soil pH is a major determinant of microbial ecosystem processes and potentially a major driver of evolution, adaptation, and diversity of ammonia oxidizers, which control soil nitrification. Archaea are major components of soil microbial communities and contribute significantly to ammonia oxidation in some soils. To determine whether pH drives evolutionary adaptation and community structure of soil archaeal ammonia oxidizers, sequences of amoA, a key functional gene of ammonia oxidation, were examined in soils at global, regional, and local scales. Globally distributed database sequences clustered into 18 well-supported phylogenetic lineages that dominated specific soil pH ranges classified as acidic (pH <5), acido-neutral (5 ≤ pH <7), or alkalinophilic (pH ≥ 7). To determine whether patterns were reproduced at regional and local scales, amoA gene fragments were amplified from DNA extracted from 47 soils in the United Kingdom (pH 3.5-8.7), including a pH-gradient formed by seven soils at a single site (pH 4.5-7.5). High-throughput sequencing and analysis of amoA gene fragments identified an additional, previously undiscovered phylogenetic lineage and revealed similar pH-associated distribution patterns at global, regional, and local scales, which were most evident for the five most abundant clusters. Archaeal amoA abundance and diversity increased with soil pH, which was the only physicochemical characteristic measured that significantly influenced community structure. These results suggest evolution based on specific adaptations to soil pH and niche specialization, resulting in a global distribution of archaeal lineages that have important consequences for soil ecosystem function and nitrogen cycling.

Investigation of Microbial Populations in the Extremely Metal-Contaminated Coeur d'Alene River Sediments

The deposition of mine tailings generated from 125 years of sulfidic ore mining resulted in the enrichment of Coeur d'Alene River (CdAR) sediments with significant amounts of toxic heavy metals. A review of literature suggests that microbial populations play a pivotal role in the biogeochemical cycling of elements in such mining-impacted sedimentary environments. To assess the indigenous microbial communities associated with metal-enriched sediments of the CdAR, high-density 16S microarray (PhyloChip) and clone libraries specific to bacteria (16S rRNA), ammonia oxidizers (amoA), and methanogens (mcrA) were analyzed. PhyloChip analysis provided a comprehensive assessment of bacterial populations and detected the largest number of phylotypes in Proteobacteria followed by Firmicutes and Actinobacteria. Furthermore, PhyloChip and clone libraries displayed considerable metabolic diversity in indigenous microbial populations by capturing several chemolithotrophic groups such as ammonia oxidizers, iron-reducers and -oxidizers, methanogens, and sulfate-reducers in the CdAR sediments. Twenty-two phylotypes detected on PhyloChip could not be classified even at phylum level thus suggesting the presence of novel microbial populations in the CdAR sediments. Clone libraries demonstrated very limited diversity of ammonia oxidizers and methanogens in the CdAR sediments as evidenced by the fact that only Nitrosospira- and Methanosarcina-related phylotypes were retrieved in amoA and mcrA clone libraries, respectively.

Hydrolase activities, microbial biomass and bacterial community in a soil after long-term amendment with different composts

The use of composts in agricultural soils is a widespread practice and the positive effects on soil and plants are known from numerous studies. However, there have been few attempts to compare the effects of different kinds of composts in one single study. The aim of this paper is to investigate to what extent and to which soil depth four major types of composts would affect the soil and its microbiota. In a crop-rotation field experiment, composts produced from (i) urban organic wastes, (ii) green wastes, (iii) manure and (iv) sewage sludge were applied at a rate equivalent to 175 kg N ha −1 yr −1 for 12 years. General (total organic C ( C org), total N ( N t), microbial biomass C ( C mic), and basal respiration), specific (enzyme activities related to C, N and P cycles), biochemical properties and bacterial genetic diversity (based on DGGE analysis of 16S rDNA) were analyzed at different depths (0–10, 10–20 and 20–30 cm). Compost treatment increased C org at all depths from 11 g kg −1 for control soil to 16.7 g kg −1 for the case of sewage sludge compost. Total N increased with compost treatment at 0–10 cm and 10–20 cm depths, but not at 20–30 cm. Basal respiration and C mic declined with depth, and the composts resulted in an increase of C mic and basal respiration. Enzyme activities were different depend on the enzyme and among compost treatments, but in general, the enzyme activities were higher in the upper layers (0–10 and 10–20 cm) than in the 20–30 cm layer. Diversity of ammonia oxidizers and bacteria was lower in the control than in the compost soils. The type of compost had less influence on the composition of the microbial communities than did soil depth. Some of the properties were sensitive enough to distinguish between different compost, while others were not. This stresses the need of multi-parameter approaches when investigating treatment effects on the soil microbial community. In general, with respect to measures of activity, biomass and community diversity, differences down the soil profile were more pronounced than those due to the compost treatments.

Nitrification in sequencing biofilm batch reactors: lessons from molecular approaches

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

Combining fluorescent in situ hybridization (fish) with cultivation and mathematical modeling to study population structure and function of ammonia-oxidizing bacteria in activated sludge

16S rRNA-targeted oligonucleotide probes for phylogenetically defined groups of autotrophic ammonia-oxidizing bacteria were used for analyzing the natural diversity of nitrifiers in an industrial sewage treatment plant receiving sewage with high ammonia concentrations. In this facility discontinuous aeration is used to allow for complete nitrification and denitrification. In situ hybridization revealed a yet undescribed diversity of ammonia oxidizers occurring in the plant. Surprisingly, the majority of the ammonia oxidizers were detected with probe combinations which indicate a close affiliation of these cells with Nitrosococcus mobilis. In addition, low numbers of ammonia-oxidizers related to the Nitrosomonas europaea - Nitrosomonas eutropha cluster were present. Interestingly, we also observed hybridization patterns which suggested the occurrence of a novel population of ammonia oxidizers. Confocal laser scanning microscopy revealed that all specifically stained ammonia oxidizers were clustered in microcolonies formed by rod-shaped bacteria. Combination of FISH and mathematical modeling was used to investigate diffusion limitation of ammonia and O2 within these aggregates. Model simulations suggest that mass transfer limitations inside the clusters are not as significant as the substrate limitations due to the activity of surrounding heterotrophic bacteria. To learn more about the ammonia-oxidizers of the industrial plant, we enriched and isolated ammonia-oxidizing bacteria from the activated sludge by combining classical cultivation techniques and FISH. Monitoring the isolates with the nested probe set allowed us to specifically identify those ammonia oxidizers which were found in situ to be numerically dominant. The phylogenetic relationship of these isolates determined by comparative 16S rDNA sequence analysis confirmed the affiliation suggested by FISH.

Combining fluorescent in situ hybridization (fish) with cultivation and mathematical modeling to study population structure and function of ammonia-oxidizing bacteria in activated sludge

16S rRNA-targeted oligonucleotide probes for phylogenetically defined groups of autotrophic ammonia-oxidizing bacteria were used for analyzing the natural diversity of nitrifiers in an industrial sewage treatment plant receiving sewage with high ammonia concentrations. In this facility discontinuous aeration is used to allow for complete nitrification and denitrification. In situ hybridization revealed a yet undescribed diversity of ammonia oxidizers occurring in the plant. Surprisingly, the majority of the ammonia oxidizers were detected with probe combinations which indicate a close affiliation of these cells with Nitrosococcus mobilis. In addition, low numbers of ammonia-oxidizers related to the Nitrosomonas europaea - Nitrosomonas eutropha cluster were present. Interestingly, we also observed hybridization patterns which suggested the occurrence of a novel population of ammonia oxidizers. Confocal laser scanning microscopy revealed that all specifically stained ammonia oxidizers were clustered in microcolonies formed by rod-shaped bacteria. Combination of FISH and mathematical modeling was used to investigate diffusion limitation of ammonia and O2 within these aggregates. Model simulations suggest that mass transfer limitations inside the clusters arc not as significant as the substrate limitations due to the activity of surrounding heterotrophic bacteria. To learn more about the ammonia-oxidizers of the industrial plant, we enriched and isolated ammonia-oxidizing bacteria from the activated sludge by combining classical cultivation techniques and FISH. Monitoring the isolates with the nested probe set allowed us to specifically identify those ammonia oxidizers which were found in situ to be numerically dominant. The phylogenetic relationship of these isolates determined by comparative 165 rDNA sequence analysts confirmed the affiliation suggested by FISH.