Monoclonal antibodies recognizing nitrite oxidoreductase of Nitrobacter hamburgensis, N. winogradskyi, and N. vulgaris.

Three monoclonal antibodies (MAbs) against nitrite oxidoreductase (NOR) of Nitrobacter hamburgensis were produced. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting analysis of the purified enzyme showed that the MAbs named Hyb 153.1 and Hyb 153.3 both recognized a protein with a molecular mass of 64,000 Da, while Hyb 153.2 recognized a protein with a molecular mass of 115,000 Da. The molecular masses of these proteins are in the same range as those of the proteins of the alpha (115,000-Da) or beta (65,000-Da) subunit of the NOR. By using the antibodies, the amount of NOR was shown to be dependent on the growth conditions. The highest level of NOR was observed in N. hamburgensis when cells were growing mixotrophically. Analysis of whole-cell extracts of N. hamburgensis, N. winogradskyi, and N. vulgaris indicated serological homology of the NORs from these species of the genus Nitrobacter. The immunological analysis enables detection of the key enzyme of the genus Nitrobacter.     

Fatty acid profiles of nitrite-oxidizing bacteria reflect their phylogenetic heterogeneity

The fatty acid profiles of all described species of the nitrite-oxidizing genera Nitrobacter, Nitrococcus, Nitrospina and Nitrospira were analyzed. The four genera had distinct profiles, which can be used for the differentiation and allocation of new isolates to these genera. The genus Nitrobacter is characterized by vaccenic acid as the main compound with up to 92% of the fatty acids and the absence of hydroxy fatty acids. The genus Nitrococcus showed cis-9-hexadecenoic acid, hexadecanoic acid and vaccenic acid as main parts. Small amounts of 3-hydroxy-dodecanoic acid were detected. The genus Nitrospina possessed tetradecanoic acid and cis-9-hcxadecenoic acid as main compounds, also 3-hydroxy-hexadecanoic acid was detected for this genus. The genus Nitrospira showed a pattern with more variations among the two described species. These organisms are characterized by the cis-7 and cis-11-isomers of hexadecenoic acid. For Nitrospira moscoviensis a specific new fatty acid was found, which represented the major constituent in the fatty acid profiles of autotrophically grown cultures. It was identified as 11-methyl-hexadecanoic acid. Since this compound is not known for other bacterial taxa, it represents a potential lipid marker for the detection of Nitrospira moscoviensis relatives in enrichment cultures and environmental samples. A cluster analysis of the fatty acid profiles is in accordance with 16S rRNA sequence-based phylogeny of the nitrite-oxidizing bacteria.     

Microenvironments and distribution of nitrifying bacteria in a membrane-bound biofilm

The distribution of nitrifying bacteria of the genera Nitrosomonas, Nitrosospira, Nitrobacter and Nitrospira was investigated in a membrane-bound biofilm system with opposed supply of oxygen and ammonium. Gradients of oxygen, pH, nitrite and nitrate were determined by means of microsensors while the nitrifying populations along these gradients were identified and quantified using fluorescence in situ hybridization (FISH) in combination with confocal laser scanning microscopy. The oxic part of the biofilm which was subjected to high ammonium and nitrite concentrations was dominated by Nitrosomonas europaea -like ammonia oxidizers and by members of the genus Nitrobacter. Cell numbers of Nitrosospira sp. were 1–2 orders of magnitude lower than those of N. europaea . Nitrospira sp. were virtually absent in this part of the biofilm, whereas they were most abundant at the oxic–anoxic interface. In the totally anoxic part of the biofilm, cell numbers of all nitrifiers were relatively low. These observations support the hypothesis that N. europaea and Nitrobacter sp. can out-compete Nitrosospira and Nitrospira spp. at high substrate and oxygen concentrations. Additionally, they suggest microaerophilic behaviour of yet uncultured Nitrospira sp. as a factor of its environmental competitiveness.     

Isolation and immunocytochemical location of the nitrite-oxidizing system in Nitrospira moscoviensis

A membrane-associated nitrite-oxidizing system of Nitrospira moscoviensis was isolated from heat-treated membranes. The four major proteins of the enzyme fraction had apparent molecular masses of 130, 62, 46, and 29 kDa, respectively. The nitrite-oxidizing activity was dependent on the presence of molybdenum. In contrast to the nitrite oxidoreductase of Nitrobacter hamburgensis X14, the activity of the nitrite-oxidizing system of Ns. moscoviensis increased when solubilized by heat treatment. Electron microscopy of the purified enzyme revealed uniform particles with a size of approximately 7 × 9 nm. SDS-immunoblotting analysis of crude extracts showed that the monoclonal antibodies Hyb 153–3, which recognize the β-subunit of the nitrite oxidoreductase from Nitrobacter, reacted with a protein of 50 kDa in Ns. moscoviensis. This protein corresponded to the protein of 46 kDa of the purified enzyme and contained a b-type cytochrome. Using electron microscopic immunocytochemistry and the monoclonal antibodies Hyb 153–3, the nitrite-oxidizing system of Ns. moscoviensis was shown to be located in the periplasmic space. Here a periodic arrangement of membrane-associated particles was found on the outside of the cytoplasmic membrane in the form of a hexagonal pattern. It is supposed that these particles represent the nitrite-oxidizing system in Nitrospira. Received: 22 August 1997 / Accepted: 1 November 1997     

Development and application of a PCR-denaturing gradient gel electrophoresis tool to study the diversity of Nitrobacter-like nxrA sequences in soil

A new PCR-denaturing gel gradient electrophoresis (DGGE) tool based on the functional gene nxrA encoding the catalytic subunit of the nitrite oxidoreductase in nitrite-oxidizing bacteria (NOB) has been developed. The first aim was to determine if the primers could target representatives of NOB genera: Nitrococcus and Nitrospira. The primers successfully amplified nxrA gene sequences from Nitrococcus mobilis, but not from Nitrospira marina. The second aim was to develop a PCR-DGGE tool to characterize NOB community structure on the basis of Nitrobacter-like partial nrxA gene sequences (Nb-nxrA). We tested (1) the ability of this tool to discriminate between Nitrobacter strains, and (2) its ability to reveal changes in the community structure of NOB harbouring Nb-nrxA sequences induced by light grazing or intensive grazing in grassland soils. The DGGE profiles clearly differed between the four Nitrobacter strains tested. Differences in the structure of NOB community were revealed between grazing regimes. Phylogenetic analysis of the sequences corresponding to different DGGE bands showed that Nb-nxrA sequences did not group in management-specific clusters. Most of the nxrA sequences obtained from soils differed from nxrA sequences of NOB strains. Along with existing tools for characterizing the community structure of nitrifiers, this new approach is a significant step forward to performing comprehensive studies on nitrification.     

Immunological Detection of Nitrospira-like Bacteria in Various Soils

Chemolithotrophic nitrite oxidizers were enriched from five different soils including freshwater marsh, permafrost, garden, agricultural, and desert soils and monitored during the cultivation procedure. Immunoblot analysis was used to identify the nitrite oxidizing organisms with monoclonal antibodies, which recognize the key enzyme of nitrite oxidation in a genus-specific reaction [Bartosch et al. (1999) Appl Environ Microbiol 65:4126-4133]. The morphological characteristics of the enriched nitrite oxidizers were additionally studied using transmission electron microscopy (TEM) and fluorescence microscopy. By means of the antibodies and TEM analysis Nitrospira could be clearly identified in enrichment cultures derived from freshwater marsh and from permafrost soil. Nitrospira cells were enriched simultaneously with cells of the genus Nitrobacter when nitrite concentrations of 0.2 g of NaNO2 L(-1) were used. However, in enrichment cultures containing 2 g of NaNO2 L(-1) Nitrobacter was exclusively detected. During fluorescence microscopic observations of DAPI stained samples microcolonies were found in enrichment cultures from freshwater marsh, permafrost, garden, and agricultural soil. They had a similar morphology to Nitrospira-like microcolonies from activated sludge. In conclusion, Nitrospira seems to be not only a common aquatic but also a usual soil bacterium.     

Nitrite concentration influences the population structure of Nitrospira-like bacteria

Chemolithoautotrophic nitrite oxidizers of the genus Nitrospira are a monophyletic but diverse group of organisms, are widely distributed in many natural habitats, and play a key role in nitrogen elimination during biological wastewater treatment. Phylogenetic analyses of cloned 16S rRNA genes and fluorescence in situ hybridization with newly developed rRNA-targeted oligonucleotide probes revealed coexistence of uncultured members of sublineages I and II of the genus Nitrospira in biofilm and activated sludge samples taken from nitrifying wastewater treatment plants. Quantitative microscopic analyses of their spatial arrangement relative to ammonia oxidizers in the biofilm and activated sludge flocs showed that members of the Nitrospira sublineage I occurred significantly more often in immediate vicinity to ammonia oxidizers than would be expected from random community assembly while such a relationship was not observed for Nitrospira sublineage II. This spatial distribution suggested a niche differentiation of these coexisting Nitrospira populations with respect to their preferred concentrations of nitrite. This hypothesis was tested by mathematical modelling of nitrite consumption and resulting nitrite gradients in nitrifying biofilms and by quantifying the abundance of sublineage I and II Nitrospira in activated sludge during incubations with nitrite in different concentrations. Consistent with the observed localization patterns, a higher nitrite concentration selected for sublineage I but suppressed sublineage II Nitrospira.     

Comparison of the Morphology and Deoxyribonucleic Acid Composition of 27 Strains of Nitrifying Bacteria

The gross morphology, fine structure, and per cent guanine plus cytosine (GC) composition of deoxyribonucleic acid of 27 strains of nitrifying bacteria were compared. Based on morphological differences, the ammonia-oxidizing bacteria were separated into four genera. Nitrosomonas species and Nitrosocystis species formed one homogenous group, and Nitrosolobus species and Nitrosospira species formed a second homogenous group in respect to their deoxyribonucleic acid GC compositions. Similarly, the nitrite-oxidizing bacteria were separated into three genera based on their morphology. The members of two of these nitrite-oxidizing genera, Nitrobacter and Nitrococcus, had similar GC compositions, but Nitrospina gracilis had a significantly lower GC composition than the members of the other two genera.     

Evolutionary relationships among ammonia- and nitrite-oxidizing bacteria.

Comparative 16S rRNA sequencing was used to evaluate phylogenetic relationships among selected strains of ammonia- and nitrite-oxidizing bacteria. All characterized strains were shown to be affiliated with the proteobacteria. The study extended recent 16S rRNA-based studies of phylogenetic diversity among nitrifiers by the comparison of eight strains of the genus Nitrobacter and representatives of the genera Nitrospira and Nitrospina. The later genera were shown to be affiliated with the delta subdivision of the proteobacteria but did not share a specific relationship to each other or to other members of the delta subdivision. All characterized Nitrobacter strains constituted a closely related assemblage within the alpha subdivision of the proteobacteria. As previously observed, all ammonia-oxidizing genera except Nitrosococcus oceanus constitute a monophyletic assemblage within the beta subdivision of the proteobacteria. Errors in the 16S rRNA sequences for two strains previously deposited in the databases by other investigators (Nitrosolobus multiformis C-71 and Nitrospira briensis C-128) were corrected. Consideration of physiology and phylogenetic distribution suggested that nitrite-oxidizing bacteria of the alpha and gamma subdivisions are derived from immediate photosynthetic ancestry. Each nitrifier retains the general structural features of the specific ancestor's photosynthetic membrane complex. Thus, the nitrifiers, as a group, apparently are not derived from an ancestral nitrifying phenotype.     

In situ characterization of nitrifiers in an activated sludge plant: detection of Nitrobacter Spp

Aims: The purpose of this work was to investigate microbial ecology of nitrifiers at the genus level in a typical full-scale activated sludge plant. Methods and Results: Grab samples of mixed liquor were collected from a plug-flow reactor receiving domestic wastewater. Fluorescent in situ hybridization technique (FISH) was used to characterize both ammonia oxidizing bacteria (AOB) and nitrite oxidizing bacteria (NOB) in combination with Confocal Scanning Laser Microscope (CSLM). Fluorescently labelled, 16S rRNA-targeted oligonucleotide probes were used in this study. Both Nitrosomonas and Nitrosospira genera as AOB and Nitrobacter and Nitrospira genera as NOB were sought with genus specific probes Nsm156, Nsv443 and NIT3 and NSR1156, respectively. Conclusions: It was shown that Nitrosospira genus was dominant in the activated sludge system studied, although Nitrosomonas is usually assumed to be the dominant genus. At the same time, Nitrobacter genus was detected in activated sludge samples. Significance and Impact of the Study: Previous studies based on laboratory scale pilot plants employing synthetic wastewater suggested that only Nitrospira are found in wastewater treatment plants. We have shown that Nitrobacter genus might also be present. We think that these kinds of studies may not give a valid indication of the microbial diversity of the real full-scale plants fed with domestic wastewater.     

Nitrospira-like bacteria associated with nitrite oxidation in freshwater aquaria

Oxidation of nitrite to nitrate in aquaria is typically attributed to bacteria belonging to the genus Nitrobacter which are members of the alpha subdivision of the class Proteobacteria. In order to identify bacteria responsible for nitrite oxidation in aquaria, clone libraries of rRNA genes were developed from biofilms of several freshwater aquaria. Analysis of the rDNA libraries, along with results from denaturing gradient gel electrophoresis (DGGE) on frequently sampled biofilms, indicated the presence of putative nitrite-oxidizing bacteria closely related to other members of the genus Nitrospira. Nucleic acid hybridization experiments with rRNA from biofilms of freshwater aquaria demonstrated that Nitrospira-like rRNA comprised nearly 5% of the rRNA extracted from the biofilms during the establishment of nitrification. Nitrite-oxidizing bacteria belonging to the alpha subdivision of the class Proteobacteria (e.g., Nitrobacter spp.) were not detected in these samples. Aquaria which received a commercial preparation containing Nitrobacter species did not show evidence of Nitrobacter growth and development but did develop substantial populations of Nitrospira-like species. Time series analysis of rDNA phylotypes on aquaria biofilms by DGGE, combined with nitrite and nitrate analysis, showed a correspondence between the appearance of Nitrospira-like bacterial ribosomal DNA and the initiation of nitrite oxidation. In total, the data suggest that Nitrobacter winogradskyi and close relatives were not the dominant nitrite-oxidizing bacteria in freshwater aquaria. Instead, nitrite oxidation in freshwater aquaria appeared to be mediated by bacteria closely related to Nitrospira moscoviensis and Nitrospira marina.     

Use of polyclonal antibodies to detect and quantify the NOR protein of nitrite oxidizers in complex environments

In the approaches or models which aim to understand and/or predict how the functioning of ecosystems may be affected by perturbations or disturbances, little attention is generally given to microorganisms. Even when they are taken into account as indicators, variables which are poorly informative about the changes in the microbial functioning (microbial biomass or diversity or total number of microorganisms) are often used. To be able to estimate, in complex environments, the quantity of enzymes involved in key ecosystem processes may constitute a useful complementary tool. Here, we describe an immunological method for detecting and quantifying, in complex environments, the nitrite oxidoreductase (NOR), responsible for the oxidation of nitrite to nitrate. The alpha-catalytic subunit of the enzyme was purified from Nitrobacter hamburgensis and used for the production of polyclonal antibodies. These antibodies were used to detect and quantify the NOR by a chemifluorescence technique on Western blots after separation of total proteins from pure cultures and soil samples. They recognized the alpha-NOR of all the Nitrobacter species described to date, but no reaction was observed with members of other nitrite-oxidizing genera. The detection threshold and reproducibility of the proposed method were evaluated. The feasibility of its use to quantify NOR in a soil was tested.     

Competition between Nitrospira spp. and Nitrobacter spp. in nitrite-oxidizing bioreactors

In this work the question was addressed if in nitrite-oxidizing activated sludge systems the environmental competition between Nitrobacter spp. and Nitrospira spp., which only recently has been discovered to play a role in these systems, is affected by the nitrite concentrations. Two parallel chemostats were inoculated with nitrifying-activated sludge containing Nitrospira and operated under identical conditions. After addition of Nitrobacter to both chemostats, the nitrite concentration in the influent of one of the chemostats was increased such that nitrite peaks in the bulk liquid of this reactor were detected. The other chemostat served as control reactor, which always had a constant nitrite influent concentration. The relative cellular area (RCA) of Nitrospira and Nitrobacter was determined by quantitative fluorescence in situ hybridization (FISH). The nitrite perturbation stimulated the growth of Nitrobacter while in the undisturbed control chemostat Nitrospira dominated. Overall, the results of this experimental study support the hypothesis that Nitrobacter is a superior competitor when resources are abundant, while Nitrospira thrive under conditions of resource scarcity. Interestingly, the dominance of Nitrobacter over Nitrospira, caused by the elevated nitrite concentrations, could not be reverted by lowering the available nitrite concentration to the original level. One possible explanation for this result is that when Nitrobacter is present at a certain cell density it is able to inhibit the growth of Nitrospira. An alternative explanation would be that the length of the experimental period was not long enough to observe an increase of the Nitrospira population.     

Characteristics of two marine nitrite oxidizing bacteria,Nitrospina gracilis nov. gen. nov. sp. and Nitrococcus mobilis nov. gen. nov. sp.

Summary Two new nitrite oxidizing bacteria for which the names Nitrococcus mobilis and Nitrospina gracilis are proposed were isolated from the marine environment. Nitrococcus mobilis was cultured from South Pacific waters and it is a large motile coccus with unique tubular cytomembranes. Nitrospina gracilis was isolated from South Atlantic waters and it is a long slender rod which lacks an extensive cytomembrane system. Both are obligate marine organisms and both are obligate chemoautotrophs. The fine structure of these organisms is detailed.Contribution No. 2631 from the Woods Hole Oceanographic Institution, Woods Hole, Massachusetts 02543.     

Unexpected diversity of chlorite dismutases: a catalytically efficient dimeric enzyme from Nitrobacter winogradskyi

Chlorite dismutase (Cld) is a unique heme enzyme catalyzing the conversion of ClO(2)(-) to Cl(-) and O(2). Cld is usually found in perchlorate- or chlorate-reducing bacteria but was also recently identified in a nitrite-oxidizing bacterium of the genus Nitrospira. Here we characterized a novel Cld-like protein from the chemolithoautotrophic nitrite oxidizer Nitrobacter winogradskyi which is significantly smaller than all previously known chlorite dismutases. Its three-dimensional (3D) crystal structure revealed a dimer of two identical subunits, which sharply contrasts with the penta- or hexameric structures of other chlorite dismutases. Despite a truncated N-terminal domain in each subunit, this novel enzyme turned out to be a highly efficient chlorite dismutase (K(m) = 90 μM; k(cat) = 190 s(-1); k(cat)/K(m) = 2.1 × 10(6) M(-1) s(-1)), demonstrating a greater structural and phylogenetic diversity of these enzymes than was previously known. Based on comparative analyses of Cld sequences and 3D structures, signature amino acid residues that can be employed to assess whether uncharacterized Cld-like proteins may have a high chlorite-dismutating activity were identified. Interestingly, proteins that contain all these signatures and are phylogenetically closely related to the novel-type Cld of N. winogradskyi exist in a large number of other microbes, including other nitrite oxidizers.     

Moderately thermophilic nitrifying bacteria from a hot spring of the Baikal rift zone

Samples from three hot springs (Alla, Seya and Garga) located in the northeastern part of Baikal rift zone (Buryat Republic, Russia) were screened for the presence of thermophilic nitrifying bacteria. Enrichment cultures were obtained solely from the Garga spring characterized by slightly alkaline water (pH 7.9) and an outlet temperature of 75 degrees C. The enrichment cultures of the ammonia- and nitrite oxidizers grew at temperature ranges of 27-55 and 40-60 degrees C, respectively. The temperature optimum was approximately 50 degrees C for both groups and thus they can be designated as moderate thermophiles. Ammonia oxidizers were identified with classical and immunological techniques. Representatives of the genus Nitrosomonas and Nitrosospira-like bacteria with characteristic vibroid morphology were detected. The latter were characterized by an enlarged periplasmic space, which has not been previously observed in ammonia oxidizers. Electron microscopy, denaturing gradient gel electrophoresis analyses and partial 16S rRNA gene sequencing provided evidence that the nitrite oxidizers were members of the genus Nitrospira.     

Taxon-specific content of oligonucleotide triplets in 16S rRNAs of anoxygenic phototrophic and nitrifying bacteria

Theoretical evaluation of the content of oligonucleotide triplets AAA, CCC, and UAU in 16S rRNAs of anoxygenic phototrophic bacteria (genera Chlorobium; Chloroflexus; Chromatium: Rhodopseudomonas) and nitrifying bacteria (genera Nitrosococcus, Nitrosomonas, Nitrosolobus, Nitrosovibrio, Nitrospira, Nitrospina, Nitrobacter) showed that the number of the AAA, CCC or UAU triplets in 16S rRNAs specifically corresponds to the genus and species of bacteria. The ratio of AAA and CCC triplet numbers in the sequences of 16S rRNA (AAA/CCC) of anoxygenic phototrophic bacteria was within the range of 0.61 to 2.03, and the ratio of AAA and UAU (AAA/UAU) triplet numbers in the sequence of 16S rRNA was within the range of 2.88 to 12.00. The regions of any genus within the AAA/CCC and AAA/UAU axes did not overlap. The combination of the numbers of nucleotide triplets in 16S rRNA is genus-specific character. The similar data were obtained in the study of a physiological group of nitrifying bacteria. The range of AAA/UAU ratio was from 1.8 to 9.0, and range of AAA/CCC was from 0.9 to 2.6 for this taxon. The number of triplets in 16S rRNAs of the studied taxa was genus- and species-specific character. The biological significance of these data is the evidence that not only the sequence but the number of nucleotide triplets in 16S rRNAs reflects the phylogeny of corresponding taxa.     

Unravelling the reasons for disproportion in the ratio of AOB and NOB in aerobic granular sludge

In this study, we analysed the nitrifying microbial community (ammonium-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB)) within three different aerobic granular sludge treatment systems as well as within one flocculent sludge system. Granular samples were taken from one pilot plant run on municipal wastewater as well as from two lab-scale reactors. Fluorescent in situ hybridization (FISH) and quantitative PCR (qPCR) showed that Nitrobacter was the dominant NOB in acetate-fed aerobic granules. In the conventional system, both Nitrospira and Nitrobacter were present in similar amounts. Remarkably, the NOB/AOB ratio in aerobic granular sludge was elevated but not in the conventional treatment plant suggesting that the growth of Nitrobacter within aerobic granular sludge, in particular, was partly uncoupled from the lithotrophic nitrite supply from AOB. This was supported by activity measurements which showed an approximately threefold higher nitrite oxidizing capacity than ammonium oxidizing capacity. Based on these findings, two hypotheses were considered: either Nitrobacter grew mixotrophically by acetate-dependent dissimilatory nitrate reduction (ping-pong effect) or a nitrite oxidation/nitrate reduction loop (nitrite loop) occurred in which denitrifiers reduced nitrate to nitrite supplying additional nitrite for the NOB apart from the AOB.     

Characterization of Chlamydia trachomatis antigens with monoclonal and polyclonal antibodies

We used monoclonal antibodies (MAbs) to examine the antigenic specificity and biologic function of several Chlamydia trachomatis antigens. Thirteen distinct MAbs to eight C. trachomatis antigens were produced. Six MAbs reacted with unique epitopes on the major outer membrane protein (MOMP) and two of these had neutralizing activity. MAbs were produced to each of the chlamydial antigens with molecular masses of 10, 29, 32, 57, 60, 70, and 75 kilodaltons (kDa). These MAbs showed species and genus specificity in an immunoblot assay. None of the MAbs had neutralizing activity. The epitopes recognized on MOMP, 29-, and 10-kDa (presumably lipopolysaccharide) antigens were surface exposed. MAbs to the 75-kDa, 57-kDa, and MOMP antigens were used for immunoaffinity purification of these antigens to produce monospecific antisera in mice. With polyclonal sera, we found that the 75-kDa antigen was also immunoaccessible and that antibody to MOMP and 75-kDa antigens neutralized C. trachomatis infectivity. We conclude that, in addition to MOMP and lipopolysaccharide, antigens with molecular masses of 75 and 29 kDa are surface exposed. Antibodies to MOMP and 75-kDa antigens can neutralize the organism in vitro.     

Effect of temperature and free ammonia on nitrification and nitrite accumulation in landfill leachate and analysis of its nitrifying bacterial community by FISH

The cause of seasonal failure of a nitrifying municipal landfill leachate treatment plant utilizing a fixed biofilm was investigated by wastewater analyses and batch respirometric tests at every treatment stage. Nitrification of the leachate treatment plant was severely affected by the seasonal temperature variation. High free ammonia (NH3-N) inhibited not only nitrite oxidizing bacteria (NOB) but also ammonia oxidizing bacteria (AOB). In addition, high pH also increased free ammonia concentration to inhibit nitrifying activity especially when the NH4-N level was high. The effects of temperature and free ammonia of landfill leachate on nitrification and nitrite accumulation were investigated with a semi-pilot scale biofilm airlift reactor. Nitrification rate of landfill leachate increased with temperature when free ammonia in the reactor was below the inhibition level for nitrifiers. Leachate was completely nitrified up to a load of 1.5 kg NH4-N m(-3)d(-1) at 28 degrees C. The activity of NOB was inhibited by NH3-N resulting in accumulation of nitrite. NOB activity decreased more than 50% at 0.7 mg NH3-N L(-1). Fluorescence in situ hybridization (FISH) was carried out to analyze the population of AOB and NOB in the nitrite accumulating nitrifying biofilm. NOB were located close to AOB by forming small clusters. A significant fraction of AOB identified by probe Nso1225 specifically also hybridized with the Nitrosomonas specific probe Nsm156. The main NOB were Nitrobacter and Nitrospira which were present in almost equal amounts in the biofilm as identified by simultaneous hybridization with Nitrobacter specific probe Nit3 and Nitrospira specific probe Ntspa662.