Purification of the key enzyme complexes of the anammox pathway from DEMON sludge.

Anaerobic Ammonium Oxidation ("anammox") is a bacterial process in which nitrite and ammonium are converted into nitrogen gas and water, yielding energy for the cell. Anammox is an important branch of the global biological nitrogen cycle, being responsible for up to 50% of the yearly nitrogen removal from the oceans. Strikingly, the anammox process uniquely relies on the extremely reactive and toxic compound hydrazine as a free intermediate. Given its global importance and biochemical novelty, there is considerable interest in the enzymes at the heart of the anammox pathway. Unfortunately, obtaining these enzymes in sufficiently large amounts for biochemical and structural studies is problematic, given the slow growth of pure cultures of anammox bacteria when high cell densities are required. However, the anammox process is being applied in wastewater treatment to remove nitrogenous waste in processes like DEamMONification (DEMON). In plants using such processes, which rely on a combination of aerobic ammonia-oxidizers and anammox organisms, kilogram amounts of anammox bacteria-containing sludge are readily available. Here, we report a protein isolation protocol starting from anammox cells present in DEMON sludge from a wastewater treatment plan that readily yields pure preparations of key anammox proteins in the tens of milligrams, including hydrazine synthase HZS and hydrazine dehydrogenase (HDH), as well as hydroxylamine oxidoreductase (HAO). HDH and HAO were active and of sufficient quality for biochemical studies and for HAO, the crystal structure could be determined. The method presented here provides a viable way to obtain materials for the study of proteins not only from the central anammox metabolism but also for the study of other exciting aspects of anammox bacteria, such as for example, their unusual ladderane lipids.

Abundance and diversity of nitrogen-removing microorganisms in the UASB-anammox reactor.

Anaerobic ammonium oxidation is considered to be the most economical and low-energy biological nitrogen removal process. So far, anammox bacteria have not yet been purified from cultures. Some nitrogen-removing microorganisms cooperate to perform the anammox process. The objective of this research was to analyze the abundance and diversity of nitrogen-removing microorganisms in an anammox reactor started up with bulking sludge at room temperature. In this study, the ammonia-oxidizing archaea phylum Crenarchaeota was enriched from 9.2 to 53.0%. Nitrosomonas, Nitrosococcus, and Nitrosospira, which are ammonia-oxidizing bacteria, increased from 3.2, 1.7, and 0.1% to 12.8, 20.4, and 3.3%, respectively. Ca. Brocadia, Ca. Kuenenia, and Ca. Scalindua, which are anammox bacteria, were detected in the seeding sludge, accounting for 77.1, 11.5, and 10.6%. After cultivation, the dominant genus changed to Ca. Kuenenia, accounting for 82.0%. Nitrospirae, nitrite oxidation bacteria, decreased from 2.2 to 0.1%, while denitrifying genera decreased from 12.9 to 2.1%. The results of this study contribute to the understanding of nitrogen-removing microorganisms in an anammox reactor, thereby facilitating the improvement of such reactors. However, the physiological and metabolic functions of the ammonia-oxidizing archaea community in the anammox reactor need to be investigated in further studies.

A rapid collection of yet unknown ammonia oxidizers in pure culture from activated sludge.

Nitrification is an important reaction in the biological nitrogen removal process in wastewater treatment plants (WWTPs). As ammonia-oxidizing microbes are slow-growing and sensitive to environmental factors such as free ammonia, pure strains are hard to obtain, preventing our understanding of their physiological characteristics. To conquer this hurdle, we report a high-throughput isolation technique based on scattering signatures, which exploits the tendency of many ammonia-oxidizing bacteria (AOB) to form microcolonies in activated sludge. The AOB microcolonies were directly sorted from the activated sludge without long incubation and enrichment bias, and were sequentially inoculated into 96-well microtiter plates containing growth medium. Phylogenetic analysis of the pure strains isolated in this study revealed a deeply branching and unrecognized lineage and diversity within the genus Nitrosomonas, beyond our expectation.

Evaluating the feasibility of ratio control strategy for achieving partial nitritation in a continuous floccular sludge reactor: Experimental demonstration.

To investigate the applicability of ratio control strategy to other systems, a continuous floccular sludge reactor was used in this study. It was found that nitrite accumulation was barely detected throughout 70days' investigation, being the average concentration in the effluent of 0.7±0.4mg/L. Batch experiments indicated that low dissolved oxygen (DO<0.3mg·L-1) greatly repressed the ammonium oxidizing bacteria (AOB) but only slightly inhibited the nitrite oxidizing bacteria (NOB). However, high-throughput sequencing revealed that the ratio of abundance between Nitrospira and Nitrosomonas, being the dominant NOB and AOB respectively, was considerably low (1.2%/18.7%). The weak oxygen gradients in floccular sludge and the selectively enriched K-strategist NOB Nitrospira under oxygen-limited conditions were both contributed to the failure of achieving partial nitritation; therefore, the rapid start-up of partial nitritation process based on proposed ratio control strategy is not feasible for continuous floccular sludge systems treating low-strength wastewater.

Nitrosomonas stercoris sp. nov., a Chemoautotrophic Ammonia-Oxidizing Bacterium Tolerant of High Ammonium Isolated from Composted Cattle Manure.

Among ammonia-oxidizing bacteria, Nitrosomonas eutropha-like microbes are distributed in strongly eutrophic environments such as wastewater treatment plants and animal manure. In the present study, we isolated an ammonia-oxidizing bacterium tolerant of high ammonium levels, designated strain KYUHI-S(T), from composted cattle manure. Unlike the other known Nitrosomonas species, this isolate grew at 1,000 mM ammonium. Phylogenetic analyses based on 16S rRNA and amoA genes indicated that the isolate belonged to the genus Nitrosomonas and formed a unique cluster with the uncultured ammonia oxidizers found in wastewater systems and animal manure composts, suggesting that these ammonia oxidizers contributed to removing higher concentrations of ammonia in strongly eutrophic environments. Based on the physiological and phylogenetic data presented here, we propose and call for the validation of the provisional taxonomic assignment Nitrosomonas stercoris, with strain KYUHI-S as the type strain (type strain KYUHI-S(T) = NBRC 110753(T) = ATCC BAA-2718(T)).

A framework for establishing predictive relationships between specific bacterial 16S rRNA sequence abundances and biotransformation rates.

The rates at which wastewater treatment plant (WWTP) microbial communities biotransform specific substrates can differ by orders of magnitude among WWTP communities. Differences in taxonomic compositions among WWTP communities may predict differences in the rates of some types of biotransformations. In this work, we present a novel framework for establishing predictive relationships between specific bacterial 16S rRNA sequence abundances and biotransformation rates. We selected ten WWTPs with substantial variation in their environmental and operational metrics and measured the in situ ammonia biotransformation rate constants in nine of them. We isolated total RNA from samples from each WWTP and analyzed 16S rRNA sequence reads. We then developed multivariate models between the measured abundances of specific bacterial 16S rRNA sequence reads and the ammonia biotransformation rate constants. We constructed model scenarios that systematically explored the effects of model regularization, model linearity and non-linearity, and aggregation of 16S rRNA sequences into operational taxonomic units (OTUs) as a function of sequence dissimilarity threshold (SDT). A large percentage (greater than 80%) of model scenarios resulted in well-performing and significant models at intermediate SDTs of 0.13-0.14 and 0.26. The 16S rRNA sequences consistently selected into the well-performing and significant models at those SDTs were classified as Nitrosomonas and Nitrospira groups. We then extend the framework by applying it to the biotransformation rate constants of ten micropollutants measured in batch reactors seeded with the ten WWTP communities. We identified phylogenetic groups that were robustly selected into all well-performing and significant models constructed with biotransformation rates of isoproturon, propachlor, ranitidine, and venlafaxine. These phylogenetic groups can be used as predictive biomarkers of WWTP microbial community activity towards these specific micropollutants. This work is an important step towards developing tools to predict biotransformation rates in WWTPs based on taxonomic composition.

Effect of ammonium nitrogen concentration on the ammonia-oxidizing bacteria community in a membrane bioreactor for the treatment of anaerobically digested swine wastewater.

A membrane bioreactor (MBR) was developed for the treatment of anaerobically digested swine wastewater and to investigate the effect of ammonium nitrogen concentration on biological nitrogen removal and ammonia-oxidizing bacteria (AOB) community structures. The MBR achieved a high NH4(+)-N removal efficiency of 0.08 kgNMLSS(-1)d(-1) and removed 95% of the influent NH4(+)-N. The TN removal rate was highest of 82.62% at COD/TN and BOD5/TN ratios of 8.76 ± 0.30 and 3.02 ± 0.09, respectively. With the decrease in ammonium nitrogen concentrations, the diversity of the AOB community declined and showed a simple pattern of DGGE. However, the AOB population size remained high, with abundance of 10(7)-10(9) copies mL(-1). With the decrease of ammonium nitrogen concentrations, Nitrosomonas eutropha gradually disappeared, whereas Nitrosomonas sp. OZK11 showed constant adaptability to survive during each treatment stage. The selective effect of ammonium concentration on AOB species could be due to the affinity for NH4(+)-N. In this study, the changes of ammonium nitrogen concentrations in digested swine wastewater were found to have selective effects on the composition of AOB community, and biological nitrogen removal was improved by optimising the influencing parameters.

Isolation and characterization of a thermotolerant ammonia-oxidizing bacterium Nitrosomonas sp. JPCCT2 from a thermal power station.

A thermotolerant ammonia-oxidizing bacterium strain JPCCT2 was isolated from activated sludge in a thermal power station. Cells of JPCCT2 are short non-motile rods or ellipsoidal. Molecular phylogenetic analysis of 16S rRNA gene sequences demonstrated that JPCCT2 belongs to the genus Nitrosomonas with the highest similarity to Nitrosomonas nitrosa Nm90 (100%), Nitrosomonas sp. Nm148 (99.7%), and Nitrosomonas communis Nm2 (97.7%). However, G+C content of JPCCT2 DNA was 49.1 mol% and clearly different from N. nitrosa Nm90, 47.9%. JPCCT2 was capable of growing at temperatures up to 48 °C, while N. nitrosa Nm90 and N. communis Nm2 could not grow at 42°C. Moreover, JPCCT2 grew similarly at concentrations of carbonate 0 and 5 gL(-1). This is the first report that Nitrosomonas bacterium is capable of growing at temperatures higher than 37°C.

Minimization of nitrous oxide emission from anoxic-oxic biological nitrogen removal process: effect of influent COD/NH4+ ratio and feeding strategy.

Nitrous oxide (N(2)O) is an important greenhouse gas and biological nitrogen removal process of wastewater treatment plant is one of its sources. Mechanisms of N(2)O emissions from anoxic-oxic biological nitrogen removal process were investigated and minimizations of N(2)O emissions were carried out from the aspect of organic carbon supplement, i.e., influent COD/NH4+ ratio (C/N ratio) and feeding strategy. Results showed that during anoxic-oxic biological nitrogen removal process, most of the N(2)O emissions occurred during the oxic phase, and both nitrifier denitrification and aerobic hydroxylamine oxidation pathways were possible mechanisms responsible for N(2)O emissions. N(2)O conversion rate decreased from 6.0% to 1.3% when the influent C/N ratio was increased from 7.5 to 14.5. This was mainly because of decrease in the abundance of Nitrosomonas-like ammonia oxidizing bacteria. Step feeding and external carbon source addition could reduce N(2)O conversion rate by 66.6% and 12.0%, respectively. Both of them were feasible methods for minimizing N(2)O emission from wastewater treatment process. The low N(2)O emission of step feeding was because of its high dissolved oxygen (DO) and low ammonium concentrations during the oxic phase, while the minimization effect of external carbon source addition was ascribed to its high nitrogen removal efficiency.

Reactor performance in terms of COD and nitrogen removal and bacterial community structure of a three-stage rotating bioelectrochemical contactor.

Integrating microbial fuel cell (MFC) into rotating biological contactor (RBC) creates an opportunity for enhanced removal of COD and nitrogen coupled with energy generation from wastewater. In this study, a three-stage rotating bioelectrochemical contactor (referred to as RBC-MFC unit) integrating MFC with RBC technology was constructed for simultaneous removal of carbonaceous and nitrogenous compounds and electricity generation from a synthetic medium containing acetate and ammonium. The performance of the RBC-MFC unit was compared to a control reactor (referred to as RBC unit) that was operated under the same conditions but without current generation (i.e. open-circuit mode). The effect of hydraulic loading rate (HLR) and COD/N ratio on the performance of the two units was investigated. At low (3.05 gCOD g⁻¹N) and high COD/N ratio (6.64 gCOD g⁻¹N), both units achieved almost similar COD and ammonia-nitrogen removal. However, the RBC-MFC unit achieved significantly higher denitrification and nitrogen removal compared to the RBC unit indicating improved denitrification at the cathode due to current flow. The average voltage under 1000 Ω external resistance ranged between 0.03 and 0.30 V and between 0.02 and 0.21 V for stages 1 and 2 of the RBC-MFC unit. Pyrosequencing analysis of bacterial 16S rRNA gene revealed high bacterial diversity at the anode and cathode of both units. Genera that play a role in nitrification (Nitrospira; Nitrosomonas), denitrification (Comamonas; Thauera) and electricity generation (Geobacter) were identified at the electrodes. Geobacter was only detected on the anode of the RBC-MFC unit. Nitrifiers and denitrifiers were more abundant in the RBC-MFC unit compared to the RBC unit and were largely present on the cathode of both units suggesting that most of the nitrogen removal occurred at the cathode.

Correlating microbial community structure and composition with aeration intensity in submerged membrane bioreactors by 454 high-throughput pyrosequencing.

For understanding of the microbial community structure and composition under different aeration intensities, 454 high-throughput pyrosequencing was applied to analyze the 16S rRNA gene of bacteria in two submerged membrane bioreactors (MBRs) under low (R(L)) and high aeration (R(H)) conditions. In total, 7818 (R(L)) and 9353 (R(H)) high-quality reads were obtained, and 1230 (R(L)) and 924 (R(H)) operational taxonomic units (OTUs) were generated at 3% cutoff level, respectively. 454 pyrosequencing could also reveal the minority bacteria that were hardly detected by the conventional molecular methods. Although the core populations were shared with highly functional organization (>80%), clear differences between the samples in the two MBRs were revealed by richness-diversity indicators and Venn analyses. Notably, microbial diversity was decreased under high aeration condition, and the evolution of the populations was observed mainly in the shared OTUs. Moreover, specific comparison down to the class and genus level showed that the relative abundances of ß-Proteobacteria and γ-Proteobacteria in the R(H) community were respectively decreased by 41.5% and 66.6%, consistent with the observed membrane fouling mitigation during the reactor operation. It was also found that Nitrospira and Nitrosomonas, being nitrite oxidizing bacteria (NOB) and ammonium oxidizing bacteria (AOB), were the dominant phylogenetic groups at the genus level of both reactors, and that the high ratio of NOB to AOB populations well supported the complete ammonium oxidation performance in the two reactors. Although some populations of NOB and AOB decreased with the increase of aeration intensity, the functional stability of the nitrification process was less affected, probably due to the low influent substrate concentration and the high level of functional organization.

Characteristics of aerobic and anaerobic ammonium-oxidizing bacteria in the hyporheic zone of a contaminated river.

Both ß-proteobacterial aerobic ammonium-oxidizing bacteria (AOB) and anaerobic ammonium-oxidizing (ANAMMOX) bacteria were investigated in the hyporheic zone of a contaminated river in China containing high ammonium levels and low chemical oxygen demand. Fluorescence in-situ hybridization (FISH), denaturing gradient gel electrophoresis (DGGE) and cloning-sequencing were employed in this study. FISH analysis illustrated that AOB (average population of 3.5 %) coexisted with ANAMMOX bacteria (0.7 %). The DGGE profile revealed a high abundance and diversity of bacteria at the water-air-soil interface rather than at the water-soil interface. The redundancy analysis correlated analysis showed that the diversity of ANAMMOX bacteria was positively related to the redox potential. The newly detected sequences of ANAMMOX organisms principally belonged to the genus Candidatus "Brocadia", while most ammonia monooxygenase subunit-A gene amoA sequences were affiliated with Nitrosospira and Nitrosomonas. These results suggest that the water-air-soil interface performs an important function in the nitrogen removal process and that the bioresources of AOB and ANAMMOX bacteria can potentially be utilized for the eutrophication of rivers.

Effects of nitrogen dioxide and anoxia on global gene and protein expression in long-term continuous cultures of Nitrosomonas eutropha C91.

Nitrosomonas eutropha is an ammonia-oxidizing betaproteobacterium found in environments with high ammonium levels, such as wastewater treatment plants. The effects of NO(2) on gene and protein expression under oxic and anoxic conditions were determined by maintaining N. eutropha strain C91 in a chemostat fed with ammonium under oxic, oxic-plus-NO(2), and anoxic-plus-NO(2) culture conditions. Cells remained viable but ceased growing under anoxia; hence, the chemostat was switched from continuous to batch cultivation to retain biomass. After several weeks under each condition, biomass was harvested for total mRNA and protein isolation. Exposure of N. eutropha C91 to NO(2) under either oxic or anoxic conditions led to a decrease in proteins involved in N and C assimilation and storage and an increase in proteins involved in energy conservation, including ammonia monooxygenase (AmoCAB). Exposure to anoxia plus NO(2) resulted in increased representation of proteins and transcripts reflective of an energy-deprived state. Several proteins implicated in N-oxide metabolism were expressed and remained unchanged throughout the experiment, except for NorCB nitric oxide reductase, which was not detected in the proteome. Rather, NorY nitric oxide reductase was expressed under oxic-plus-NO(2) and anoxic-plus-NO(2) conditions. The results indicate that exposure to NO(2) results in an energy-deprived state of N. eutropha C91 and that anaerobic growth could not be supported with NO(2) as an oxidant.

Ammonia oxidizing bacterial community composition and process performance in wastewater treatment plants under low temperature conditions.

Nitrification can be difficult to maintain at wastewater treatment plants (WWTPs) during cold periods resulting in disrupted nitrogen removal. The aim of this study was to relate nitrification process performance to abundance and composition of the ammonia oxidizer communities in two closely located municipal WWTPs in Sweden during an eight month period covering seasonal changes and low temperature conditions. Both facilities showed lower NH(4)(+)-N removal efficiency and nitrification rates as temperature decreased. However, one of the plants had a more stable nitrification rate and higher ammonia removal efficiency throughout the entire period. The differences in performance was related to a shift in the composition of the bacterial ammonia oxidizing community from a Nitrosomonas oligotropha-dominated community to a mixed community including also Nitrosomonas ureae-like ammonia oxidizers. This was likely a response to differences in NH(4)(+)-N and organic loading.

Growth of ammonia-oxidizing archaea and bacteria in cattle manure compost under various temperatures and ammonia concentrations.

A recent study showed that ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing archaea (AOA) coexist in the process of cattle manure composting. To investigate their physiological characteristics, liquid cultures seeded with fermenting cattle manure compost were incubated at various temperatures (37°C, 46°C, or 60°C) and ammonium concentrations (0.5, 1, 4, or 10 mM NH (4) (+) -N). The growth rates of the AOB and AOA were monitored using real-time polymerase chain reaction analysis targeting the bacterial and archaeal ammonia monooxygenase subunit A genes. AOB grew at 37°C and 4 or 10 mM NH (4) (+) -N, whereas AOA grew at 46°C and 10 mM NH (4) (+) -N. Incubation with allylthiourea indicated that the AOB and AOA grew by oxidizing ammonia. Denaturing gradient gel electrophoresis and subsequent sequencing analyses revealed that a bacterium related to Nitrosomonas halophila and an archaeon related to Candidatus Nitrososphaera gargensis were the predominant AOB and AOA, respectively, in the seed compost and in cultures after incubation. This is the first report to demonstrate that the predominant AOA in cattle manure compost can grow and can probably oxidize ammonia under moderately thermophilic conditions.

Analysis of ammonia-oxidizing bacteria dominating in lab-scale bioreactors with high ammonium bicarbonate loading.

The ammonia-oxidizing bacterial community (AOB) was investigated in two types of laboratory-scale bioreactors performing partial oxidation of ammonia to nitrite or nitrate at high (80 mM) to extremely high (428 mM) concentrations of ammonium bicarbonate. At all conditions, the dominant AOB was affiliated to the Nitrosomonas europaea lineage as was determined by fluorescence in situ hybridization and polymerase chain reaction in combination with denaturing gradient gel electrophoresis. Molecular analysis of the mixed populations, based on the 16S rRNA and cbbL genes, demonstrated the presence of two different phylotypes of Nitrosomonas, while microbiological analysis produced a single phylotype, represented by three different morphotypes. One of the most striking features of the AOB populations encountered in the bioreactors was the domination of highly aggregated obligate microaerophilic Nitrosomonas, with unusual cellular and colony morphology, commonly observed in nitrifying bioreactors but rarely investigated by cultural methods. The latter is probably not an adaptation to stressful conditions created by high ammonia or nitrite concentrations, but oxygen seems to be a stressful factor in these bioreactors.

Low-dissolved-oxygen nitrifying systems exploit ammonia-oxidizing bacteria with unusually high yields.

In wastewater treatment plants, nitrifying systems are usually operated with elevated levels of aeration to avoid nitrification failures. This approach contributes significantly to operational costs and the carbon footprint of nitrifying wastewater treatment processes. In this study, we tested the effect of aeration rate on nitrification by correlating ammonia oxidation rates with the structure of the ammonia-oxidizing bacterial (AOB) community and AOB abundance in four parallel continuous-flow reactors operated for 43 days. Two of the reactors were supplied with a constant airflow rate of 0.1 liter/min, while in the other two units the airflow rate was fixed at 4 liters/min. Complete nitrification was achieved in all configurations, though the dissolved oxygen (DO) concentration was only 0.5 ± 0.3 mg/liter in the low-aeration units. The data suggest that efficient performance in the low-DO units resulted from elevated AOB levels in the reactors and/or putative development of a mixotrophic AOB community. Denaturing gel electrophoresis and cloning of AOB 16S rRNA gene fragments followed by sequencing revealed that the AOB community in the low-DO systems was a subset of the community in the high-DO systems. However, in both configurations the dominant species belonged to the Nitrosomonas oligotropha lineage. Overall, the results demonstrated that complete nitrification can be achieved at low aeration in lab-scale reactors. If these findings could be extended to full-scale plants, it would be possible to minimize the operational costs and greenhouse gas emissions without risk of nitrification failure.

[Diversity and community structure of soil ammonia-oxidizing bacteria in Hulunbeier Grassland, Inner Mongolia].

By the methods of polymerase chain reaction-denaturing gradient gel electrophoresis and sequence analysis, a comparative study was conducted on the diversity and community structure of soil ammonia-oxidizing bacteria in the Filifolium sibiricum steppe, Stipa baicalensis steppe, Leymus chinensis steppe, Stipa grandis steppe, and Stipa kryrowi steppe in Hulunbeier Grassland, Inner Mongolia. A significant difference was observed in the community structure of soil ammonia-oxidizing bacteria among the five steppes, with the similarity lower than 50%. The diversity of soil ammonia-oxidizing bacteria was the highest in F. sibiricum steppe, followed by in S. baicalensis steppe, L. chinensis steppe, S. kryrowi steppe, and S. grandis steppe. In the five steppes, Nitrosospira cluster 3 was the dominant group, and the Nitrosospira cluster 1, 2, and 4 as well as Nitrosomonas were also found. The community structure of soil ammonia oxidizing bacteria in F. sibiricum steppe was most complex, while that in L. chinensis steppe and S. grandis steppe was relatively simple. Correlation analysis indicated that there existed significant positive correlations between the diversity of soil ammonia-oxidizing bacteria and the soil moisture, total nitrogen, total organic carbon, and C/N ratio (P<0.05).

The community analysis of ammonia-oxidizing bacteria in wastewater treatment plants revealed by the combination of double labeled T-RFLP and sequencing.

The functional gene of amoA, which produces the α-subunit of ammonia monooxygenase (AMO), has been analyzed to reveal the microbial community structure of ammonia-oxidizing bacteria (AOB) by culture-independent methods. In this study, the distribution of the amoA gene in 10 wastewater treatment plants (WWTPs) was revealed by the fingerprinting method of terminal restriction fragment length polymorphism (T-RFLP) and comparative sequencing. T-RFLP showed diverse communities of AOB in the modified Ludzack-Ettinger process, in the anaerobic-anoxic-oxic processes, in the hanging biological contactor, and in the sequencing batch reactor. In all of these environments, long solid retention time (SRT) was expected to be the critical factor for maintaining the diverse AOB community structure. Because T-RFLP does not offer sufficient information to confirm the phylogenetic information of AOB, the microbial community structures were analyzed by comparative sequencing for seven samples that were selected by the statistical categorization using principal component analysis (PCA) among 14 samples. The phylogenetic tree of 21 operational taxonomic units (OTUs) among 88 clones obtained in this study revealed that AOB of Nitrosomonas oligotropha and europaea lineages were predominant in WWTPs. Double labeled T-RFLP produced group-specific terminal restriction fragments (T-RFs) representing several groups of AOB and offered advanced resolution comparing with the single labeled T-RFLP.