Phage-host associations in a full-scale activated sludge plant during sludge bulking.

Sludge bulking, a notorious microbial issue in activated sludge plants, is always accompanied by dramatic changes in the bacterial community. Despite large numbers of phages in sludge systems, their responses to sludge bulking and phage-host associations during bulking are unknown. In this study, high-throughput sequencing of viral metagenomes and bacterial 16S rRNA genes were employed to characterize viral and bacterial communities in a sludge plant under different sludge conditions (sludge volume index (SVI) of 180, 132, and 73 ml/g). Bulking sludges (SVI > 125 ml/g) taken about 10 months apart exhibited similar bacterial and viral composition. This reflects ecological resilience of the sludge microbial community and indicates that changes in viral and bacterial populations correlate closely with each other. Overgrowth of "Candidatus Microthrix parvicella" led to filamentous bulking, but few corresponding viral genotypes were identified. In contrast, sludge viromes were characterized by numerous contigs associated with "Candidatus Accumulibacter phosphatis," suggesting an abundance of corresponding phages in the sludge viral community. Notably, while nitrifiers (mainly Nitrosomonadaceae and Nitrospiraceae) declined significantly along with sludge bulking, their corresponding viral contigs were identified more frequently and with greater abundance in the bulking viromes, implying that phage-mediated lysis might contribute to the loss of autotrophic nitrifiers under bulking conditions.

A logistic model for the remediation of filamentous bulking in a biological nutrient removal wastewater treatment plant.

Biological nutrient removal (BNR) systems across the globe frequently experience bulking and foaming episodes, which present operational challenges such as poor sludge settling due to excessive filamentous bacteria. A full-scale BNR plant treating primarily domestic wastewater was monitored over a period of 1 year to investigate filamentous bacterial growth response under various plant operating parameters. Identification of filamentous bacteria by conventional microscopy and fluorescent in situ hybridisation indicated the dominance of Eikelboom Type021N, Thiothrix spp., Eikelboom Type 1851 and Eikelboom Type 0092. A cumulative logit model (CLM) was applied to elucidate significant relationships between the filamentous bacteria and plant operational parameters. The model could predict the potential abundance of dominant filamentous bacteria in relation to wastewater treatment plant operational parameters. Data obtained from the model corroborated with previous findings on the dominance of most filaments identified, except for Type 0092, which exhibited some unique traits. With further validation, the model could be successfully applied for identifying specific parameters which could contribute towards filamentous bulking, thus, providing a useful tool for regulating specific filamentous growth in full-scale wastewater treatment plants.

The potential role of 'Candidatus Microthrix parvicella' in phosphorus removal during sludge bulking in two full-scale enhanced biological phosphorus removal plants.

We investigated the bacterial community compositions and phosphorus removal performance under sludge bulking and non-bulking conditions in two biological wastewater treatment systems (conventional A²/O (anaerobic/anoxic/aerobic) and inverted A²/O (anoxic/anaerobic/aerobic) processes) receiving the same raw wastewater. Sludge bulking resulted in significant shift in bacterial compositions from Proteobacteria dominance to Actinobacteria dominance, characterized by the significant presence of filamentous 'Candidatus Microthrix parvicella'. Quantitative real-time polymerase chain reaction (PCR) analysis revealed that the relative abundance of 'Candidatus Accumulibacter phosphatis', a key polyphosphate-accumulating organism responsible for phosphorus removal, with respect to 16s rRNA genes of total bacteria was 0.8 and 0.7%, respectively, for the conventional and inverted A²/O systems when sludge bulking occurred, which increased to 8.2 and 12.3% during the non-bulking period. However, the total phosphorus removal performance during the bulking period (2-week average: 97 ± 1 and 96 ± 1%, respectively) was not adversely affected comparable to that during the non-bulking period (2-week average: 96 ± 1 and 96 ± 1%, respectively). Neisser staining revealed the presence of large polyphosphate granules in 'Candidatus Microthrix parvicella', suggesting that this microbial group might have been responsible for phosphorus removal during the sludge bulking period when 'Candidatus Accumulibacter phosphatis' was excluded from the systems.

Genome sequence of "Candidatus Microthrix parvicella" Bio17-1, a long-chain-fatty-acid-accumulating filamentous actinobacterium from a biological wastewater treatment plant.

"Candidatus Microthrix" bacteria are deeply branching filamentous actinobacteria which occur at the water-air interface of biological wastewater treatment plants, where they are often responsible for foaming and bulking. Here, we report the first draft genome sequence of a strain from this genus: "Candidatus Microthrix parvicella" strain Bio17-1.

Analytical solution for the modeling of the natural time-dependent reduction of waterborne viruses injected into fractured aquifers.

We propose an analytical solution in order to explain the processes that determine the fate and behavior of the viruses during transport in a fractured aquifer at Salento (Italy). The calculations yield the efficiency of filtration in fractures at a site near Nardò (Southern Italy) in reducing the numbers of enteric viruses (i.e., Enteroviruses and Norovirus) in secondary municipal effluents that have been injected in the aquifer over the period 2006-2007. The model predicted, by a theoretical expression, the time-dependent rate of virus reduction, which was in good agreement with field data. The analytical solution yields the achievable "Log reduction credits" for virus reduction in wells located at the setback distances that are usually adopted in local drinking water regulations. The resulting new analytical formula for the time-dependent reduction of viruses during subsurface transport can easily be applied in health risk-based models used to forecast the spread of waterborne diseases and provides appropriate criteria (i.e., distances) needed to meet standards for the quality of drinking water derived from undisinfected groundwater.

Quantitative PCR monitoring of antibiotic resistance genes and bacterial pathogens in three European artificial groundwater recharge systems.

Aquifer recharge presents advantages for integrated water management in the anthropic cycle, namely, advanced treatment of reclaimed water and additional dilution of pollutants due to mixing with natural groundwater. Nevertheless, this practice represents a health and environmental hazard because of the presence of pathogenic microorganisms and chemical contaminants. To assess the quality of water extracted from recharged aquifers, the groundwater recharge systems in Torreele, Belgium, Sabadell, Spain, and Nardò, Italy, were investigated for fecal-contamination indicators, bacterial pathogens, and antibiotic resistance genes over the period of 1 year. Real-time quantitative PCR assays for Helicobacter pylori, Yersinia enterocolitica, and Mycobacterium avium subsp. paratuberculosis, human pathogens with long-time survival capacity in water, and for the resistance genes ermB, mecA, blaSHV-5, ampC, tetO, and vanA were adapted or developed for water samples differing in pollutant content. The resistance genes and pathogen concentrations were determined at five or six sampling points for each recharge system. In drinking and irrigation water, none of the pathogens were detected. tetO and ermB were found frequently in reclaimed water from Sabadell and Nardò. mecA was detected only once in reclaimed water from Sabadell. The three aquifer recharge systems demonstrated different capacities for removal of fecal contaminators and antibiotic resistance genes. Ultrafiltration and reverse osmosis in the Torreele plant proved to be very efficient barriers for the elimination of both contaminant types, whereas aquifer passage followed by UV treatment and chlorination at Sabadell and the fractured and permeable aquifer at Nardò posed only partial barriers for bacterial contaminants.

Comparison between kinetics of autochthonous marine bacteria in activated sludge and granular sludge systems at different salinity and SRTs.

Biological nutrient removal performances and kinetics of autochthonous marine biomass in forms of activated sludge and aerobic granular sludge were investigated under different salinity and sludge retention time (SRT). Both the biomasses, cultivated from a fish-canning wastewater, were subjected to stepwise increases in salinity (+2 gNaCl L-1), from 30 gNaCl L-1 up to 50 gNaCl L-1 with the aim to evaluate the maximum potential in withstanding salinity by the autochthonous marine biomass. Microbial marine species belonging to the genus of Cryomorphaceae and of Rhodobacteraceae were found dominant in both the systems at the maximum salinity tested (50 gNaCl L-1). The organic carbon was removed with a yield of approximately 98%, irrespective of the salinity. Similarly, nitrogen removal occurred via nitritation-denitritation and was not affected by salinity. The ammonium utilization rate and the nitrite utilization rate were approximately of 3.60 mgNH4-N gVSS-1h-1 and 10.0 mgNO2-N gVSS-1h-1, respectively, indicating a high activity of nitrifying and denitrifying bacteria. The granulation process did not provide significant improvements in the nutrients removal process likely due to the stepwise salinity increase strategy. Biomass activity and performances resulted affected by long SRT (27 days) due to salt accumulation within the activated sludge flocs and granules. In contrast, a lower SRT (14 days) favoured the discharge of the granules and flocs with higher inert content, thereby enhancing the biomass renewing. The obtained results demonstrated that the use of autochthonous-halophilic bacteria represents a valuable solution for the treatment of high-strength carbon and nitrogen saline wastewater in a wide range of salinity. Besides, the stepwise increase in salinity and the operation at low SRT enabled high metabolic activity and to avoid excessive accumulation of salt within the biomass aggregates, limiting their physical destructuration due to the increase in loosely-bound exopolymers.

Structure analysis and performance of a microbial community from a contaminated aquifer involved in the complete reductive dechlorination of 1,1,2,2-tetrachloroethane to ethene.

An anaerobic microcosm set up with aquifer material from a 1,1,2,2-tetrachloroethane (TeCA) contaminated site and amended with butyrate showed a complete TeCA dechlorination to ethene. A structure analysis of the microbial community was performed by fluorescence in situ hybridization (FISH) with already available and on purpose designed probes from sequences retrieved through 16S rDNA clone library construction. FISH was chosen as identification tool to evaluate in situ whether the retrieved sequences belong to primary bacteria responsible for the biodegradative reactions. FISH probes identified up to 80% of total bacteria and revealed the absence or the marginal presence of known TeCA degraders and the abundance of two well-known H(2)-utilizing halorespiring bacteria, Sulfurospirillum (32.4 +/- 8.6% of total bacteria) and Dehalococcoides spp. (14.8 +/- 2.8), thereby providing a strong indication of their involvement in the dechlorination processes. These results were supported by the kinetic and thermodynamic analysis which provided indications that hydrogen was the actual electron donor for TeCA dechlorination. The specific probes, developed in this study, for known dechlorinators (i.e., Geobacter, Dehalobacter, and Sulfurospirillum species) represent a valuable tool for any future in situ bioremediation study as well as a quick and specific investigation tool for tracking their distribution in the field.

Control strategy for filamentous sludge bulking: Bench-scale test and full-scale application.

Sludge bulking caused by the overgrowth of filamentous bacteria, especially Microthrix parvicella, has been observed in WWTPs worldwide during low-temperature periods. In this study, the impacts of sludge load on the in situ growth of M. parvicella and sludge settleability were first evaluated at 15 °C over a period of 500 d using a bench-scale anaerobic-anoxic-aerobic reactor fed with raw sewage from a full-scale WWTP. When the reactor was operated at a sludge load of 0.07 ±â€¯0.015 kg Chemical Oxygen Demand (COD) (kg MLSS·d)-1 for 120 d, the sludge volume index (SVI) increased gradually from 85 mL g-1 to 157 mL g-1, and the abundance of M. parvicella quantified by qPCR and FISH methods also increased from 0.42% to 4.63% and 1.56%-13.59%, respectively. When the sludge load was further reduced to 0.04 ±â€¯0.004 kg COD (kg MLSS·d)-1, the SVI value varied in a narrow range of 135-164 mL g-1 over a duration of 280 d, while the M. parvicella abundance increased to the maximum values of 10.13% (qPCR) and 18.53% (FISH), respectively. When the sludge load was increased to 0.12 ±â€¯0.016 kg COD (kg MLSS·d)-1, filamentous abundance and SVI were reduced to 1.06% (qPCR) and 105 mL g-1 within 100 d, suggesting that it might be possible to control the growth of M. parvicella by keeping the sludge load above 0.1 kg COD (kg MLSS·d)-1. The feasibility of the strategy was further validated in the same WWTP. It was found that the SVI and filamentous abundance in winter were successfully controlled for two successive years at below 120 mL g-1 and 7% (FISH), respectively, when the sludge load was maintained at 0.14 ±â€¯0.04 kg COD (kg MLSS·d)-1 by adjusting sludge discharge, proving that this sludge-load-based strategy could be an efficient approach to control filamentous bulking.

Identity, abundance and ecophysiology of filamentous Chloroflexi species present in activated sludge treatment plants.

Filamentous Chloroflexi species are often present in activated sludge wastewater treatment plants in relatively low numbers, although bulking incidences caused by Chloroflexi filaments have been observed. A new species-specific gene probe for FISH was designed and using phylum-, subdivision-, morphotype 1851- and species-specific gene probes, the abundance of Chloroflexi filaments were monitored in samples from 126 industrial wastewater treatment plants from five European countries. Chloroflexi filaments were present in 50% of the samples, although in low quantities. In most treatment plants the filaments could only be identified with phylum or subdivision probes, indicating the presence of great undescribed biodiversity. The ecophysiology of various Chloroflexi filaments was investigated by a suite of in situ methods. The experiments revealed that Chloroflexi constituted a specialized group of filamentous bacteria only active under aerobic conditions consuming primarily carbohydrates. Many exo-enzymes were excreted, e.g. chitinase, glucuronidase and galactosidase, suggesting growth on complex polysaccharides. The surface of Chloroflexi filaments appeared to be hydrophilic compared to other filaments present. These results are generally supported by physiological studies of two new isolates. Based on the results obtained in this study, the potential role of filamentous Chloroflexi species in activated sludge is discussed.

Effect of periodic feeding on substrate uptake and storage rates by a pure culture of Thiothrix (CT3 strain).

A pure culture of Thiothrix strain CT3 has been aerobically cultured under periodic acetate feeding in a Sequencing Batch Reactor (SBR) at volumetric organic load rate of 0.12gCODL(-1)d(-1). Two different culture residence times (12d or 20d) were adopted as well as two different feed frequencies (1 and 4d(-1), for each culture residence time), the volumetric organic load rate being the same under all conditions. The transient response of the microorganism to the periodic acetate feed was investigated through batch tests with biomass withdrawn from the SBR, as function of the different SBR operating conditions. In all tested conditions, a quick transient response to the acetate spike was observed with fast increase of acetate uptake rate (ranging from 71 to 247mgCODgCOD(-1)h(-1)). This transient response was mainly due to acetate storage in form of poly-hydroxybutyrate (ranging from 45% to 64% of the observed yield) whereas the growth response (i.e. increase of production rate of active biomass) generally played a minor role (ranging from 21% to 38% of the observed yield). Apart from this general trend, culture residence time as well as feed frequency had a strong impact on transient behaviour of cultured cells. The overall transient response (i.e. maximum specific substrate removal rate) increased as culture residence time decreased or as feed frequency increased. Moreover, the ratio of storage response and growth response increased as the overall transient response decreased, i.e. the storage response was preferentially maintained when cells presented a lower transient response. The ability of the cells to increase their growth rate with respect to SBR average value was the lowest under the most unfavourable conditions (residence time 20d, feed frequency 1d(-1)) and increased with the increase in maximum substrate uptake rate.

Dechlorinating ability of TCE-fed microcosms with different electron donors.

The main objective of the work presented herein is to assess the effect of different electron donors (butyric acid and methanol) on the dechlorinating activity of two microbial cultures where active methanogenic populations are present, in an effort to evaluate the importance of the electron donor selection process. The ability of each anaerobic culture to dechlorinate TCE, when enriched with either butyric acid or methanol, was verified based on the results of gas chromatography. In addition, the fluorescent in situ hybridization (FISH) and the polymerase chain reaction (PCR) methods gave positive results for the presence of Dehalococcoides spp. According to results of the batch tests conducted in this study, it appears that the selection of the electron donor for stimulating TCE dechlorination depends on microbial culture composition; therefore, the decision on the appropriate electron donor should be based on site-specific microcosm studies.

"Microthrix parvicella", a filamentous bacterium causing bulking and foaming in activated sludge systems: a review of current knowledge.

This review summarizes the microbiology and physiology of "Microthrix parvicella" and the methods of its growth control in activated sludge wastewater treatment plants. This filamentous bacterium is of high interest because of its worldwide involvement in severe bulking and foaming at wastewater treatment plants. We present a critical analysis of physiological and kinetic data on "M. parvicella" and discuss its growth and storage abilities in various environments with the aim of understanding the strategies of this organism to successfully compete with other bacteria in activated sludge. Additionally, this review elaborates on research needs for defining reliable control strategies of bulking and foaming based on key features of "M. parvicella".

Factors affecting the growth of Microthrix parvicella: Batch tests using bulking sludge as seed sludge.

Sludge bulking caused by the overgrowth of filamentous bacteria, particularly Microthrix parvicella, is one of the challenges for the stable operation of municipal wastewater treatment plants (WWTPs). The driving forces for the development of sludge bulking, however, have not been well understood because of the extremely low growth rate of M. parvicella. In this study, batch experiments were performed using bulking sludge (sludge volume index (SVI), around 185mLg-1) from a full-scale WWTP as the seed sludge to investigate the influences of carbon source, anaerobic/aerobic alternation condition and temperature on the growth of M. parvicella. The qPCR results showed that the use of oleic acid as carbon source, anaerobic/aerobic alternation treatment and low temperature (13°C) were favorable conditions for maintaining the dominance of M. parvicella in the tested activated sludge. Under these conditions, the SVI values remained at comparatively high values of 170.5mLg-1, 162.5mLg-1 and 129.5mLg-1 after operation for approximately two months, and the relative abundances of M. parvicella were 36.7%, 9.74% and 34.07%, respectively, in comparison with the initial values of 33.04%, 29.29% and 54.66%. However, the relative abundances of M. parvicella decreased to 0.86-4.44%, 0.7% and 4.94%, respectively, under the conditions of other carbon sources, aerobic-only treatment and a temperature of 20°C. The FISH analysis gave a similar result. This study was performed with mixed sludge under controlled operating conditions, which provided a valuable information for the pure culture of M. parvicella and further investigations on its physiology and metabolism.

Effect of culture residence time on substrate uptake and storage by a pure culture of Thiothrix (CT3 strain) under continuous or batch feeding.

A pure culture of the filamentous bacterium Thiothrix, strain CT3, was aerobically cultured in a chemostat under continuous acetate feeding at three different culture residence times (RT 6, 12 or 22 d) and the same volumetric organic load rate (OLR 0.12gCOD/L/d). Cells cultured at decreasing RT in the chemostat had an increasing transient response to acetate spikes in batch tests. The maximum specific acetate removal rate increased from 25 to 185mgCOD/gCOD/h, corresponding to a 1.8 to 8.1 fold higher respective steady-state rate in the chemostat. The transient response was mainly due to acetate storage in the form of poly(3-hydroxybutyrate) (PHB), whereas no growth response was observed at any RT. Interestingly, even though the storage rate also decreased as the RT increased, the storage yield increased from 0.41 to 0.50 COD/COD. This finding does not support the traditional view that storage plays a more important role as the transient response increases. The transient response of the steady-state cells was much lower than in cells cultured under periodic feeding (at 6 d RT, from 82 to 247mgCOD/gCOD/h), with the latter cells showing both storage and growth responses. On the other hand, even though steady-state cells had no growth response and their storage rate was also less, steady-state cells showed a higher storage yield than cells cultured under dynamic feeding. This suggests that in Thiothrix strain CT3, the growth response is triggered by periodic feeding, whereas the storage response is a constitutive mechanism, independent from previous acclimation to transient conditions.

Assessment of natural or enhanced in situ bioremediation at a chlorinated solvent-contaminated aquifer in Italy: a microcosm study.

A microcosm study was used to assess the potential for in situ natural or enhanced bioremediation at a chloroethane- (i.e., tetrachloroethane, TeCA) and chloroethene-contaminated (i.e., tetrachloroethene, PCE; trichloroethene, TCE) groundwater in Northern Italy. All the live microcosms were positive for dechlorination, indicating the presence of an active native dechlorinating population in the subsurface. All the tested electron donors (i.e., yeast extract, lactate, butyrate, hydrogen) promoted enhanced dechlorination of chlorinated contaminants. Lactate- and butyrate-amended microcosms performed the best, and also dechlorinated the solvents past cis-dichloroethene (cis-DCE). The microcosm bioaugmented with a PCE-dechlorinating mixed culture containing Dehalococcoides spp. dechlorinated groundwater contaminants to DCE, vinyl chloride (VC), and ethene (ETH). In conclusion, results from this microcosm study indicate the potential for enhancing full dechlorination at the contaminated site, through a proper addition of a suitable electron donor (e.g., lactate or butyrate) and/or through bioaugmentation with a Dehalococcoides-containing culture.

In situ groundwater and sediment bioremediation: barriers and perspectives at European contaminated sites.

This paper contains a critical examination of the current application of environmental biotechnologies in the field of bioremediation of contaminated groundwater and sediments. Based on analysis of conventional technologies applied in several European Countries and in the US, scientific, technical and administrative barriers and constraints which still need to be overcome for an improved exploitation of bioremediation are discussed. From this general survey, it is evident that in situ bioremediation is a highly promising and cost-effective technology for remediation of contaminated soil, groundwater and sediments. The wide metabolic diversity of microorganisms makes it applicable to an ever-increasing number of contaminants and contamination scenarios. On the other hand, in situ bioremediation is highly knowledge-intensive and its application requires a thorough understanding of the geochemistry, hydrogeology, microbiology and ecology of contaminated soils, groundwater and sediments, under both natural and engineered conditions. Hence, its potential still remains partially unexploited, largely because of a lack of general consensus and public concerns regarding the lack of effectiveness and control, poor reliability, and possible occurrence of side effects, for example accumulation of toxic metabolites and pathogens. Basic, applied and pre-normative research are all needed to overcome these barriers and make in situ bioremediation more reliable, robust and acceptable to the public, as well as economically more competitive. Research efforts should not be restricted to a deeper understanding of relevant microbial reactions, but also include their interactions with the large array of other relevant phenomena, as a function of the truly variable site-specific conditions. There is a need for a further development and application of advanced biomolecular tools for site investigation, as well as of advanced metabolic and kinetic modelling tools. These would allow a quicker evaluation of the bioremediation potential of a site, and in turn a preliminary assessment of the technical feasibility of the chosen bioprocess which could replace or at least reduce the need for time-consuming and expensive field tests. At the same time, field tests will probably remain unavoidable for a detailed design of full scale remedial actions and the above reported tools will in any event be useful for a better design and a more reliable operation.

Methods for detection and visualization of intracellular polymers stored by polyphosphate-accumulating microorganisms.

Polyphosphate-accumulating microorganisms (PAOs) are important in enhanced biological phosphorus (P) removal. Considerable effort has been devoted to understanding the biochemical nature of enhanced biological phosphorus removal (EBPR) and it has been shown that intracellular polymer storage plays an important role in PAO's metabolism. The storage capacity of PAOs gives them a competitive advantage over other microorganisms present that are not able to accumulate internal reserves. Intracellular polymers stored by PAOs include polyphosphate (poly-P), polyhydroxyalkanoates (PHAs) and glycogen. Staining procedures for qualitative visualization of polymers by optical microscopy and combinations of these procedures with molecular tools for in situ identification are described here. The strengths and weaknesses of widely used polymer quantification methods that require destruction of samples, are also discussed. Finally, the potential of in vivo nuclear magnetic resonance (NMR) spectroscopy for on-line measurement of intracellular reserves is reported.

Filamentous Alphaproteobacteria associated with bulking in industrial wastewater treatment plants.

The phylogeny and distribution of filamentous Alphaproteobacteria, morphologically similar to "Nostocoida limicola" and Eikelboom Type 021N that cause the solids separation problem of bulking in industrial activated sludge plants is described here. A combination of culture-dependent and culture-independent molecular methods has characterized 5 novel species. 16S rRNA targeted oligonucleotide probes were designed for their in situ identification by fluorescence in situ hybridisation (FISH) and used to monitor their presence in 86 WWTPs treating different industrial effluents in four European countries. The involvement of these bacteria in bulking in these plants was confirmed. Filaments hybridising with the ALF-968 probe for the Alphaproteobacteria were present in 65% of the WWTPs examined. They were dominant and therefore probably responsible for bulking in 25.5% of them. The heterogeneous filamentous alphaproteobacterial populations in these communities could be completely identified after application of the oligonucleotide probes used in this study in 91% of the plants containing them. The only filamentous Alphaproteobacteria retrieved in pure culture was isolated from three different industrial WWTPs plants. None of these isolates could grow anaerobically on glucose or denitrify, but all grew aerobically and heterotrophically on a range of carbon sources. Although morphologically similar to the Eikelboom Type 021N morphotype, they were not involved in sulphur metabolism. These bacteria accumulated lipidic storage granules that were associated with their presence under the unbalanced growth conditions existing in these plants.

Influence of aeration and sludge retention time on ammonium oxidation to nitrite and nitrate.

Partial nitrification to nitrite was reported to be technically feasible and economically favourable, especially when wastewater with high ammonium concentrations or low C/N ratios are treated. Nitritation can be obtained by selectively inhibiting nitrite oxidizing microrganisms through appropriate regulation of the system's pH, temperature, and sludge retention time. In addition to already known methods, the work showed that aeration patterns may play a relevant role too. Nitrification tests were performed in two lab-scale reactors operated under continuous and intermittent aeration, respectively. In both plants, temperature was maintained at 32 degrees C and pH was regulated at 7.2 by providing external buffer capacity when needed. The results showed that partial nitrification to nitrite was steadily obtained under oxygen limitation, independent of the sludge age. Therefore, the aeration pattern is proposed as an alternative parameter to the sludge retention time for controlling ammonium oxidation to nitrite.